新型可复制型抗肿瘤DNA疫苗PSCK-2PFcGB的抑瘤活性及免疫学机制研究
|
摘要
随着免疫学、分子生物学和细胞生物学理论与技术的不断深入发展，更高效、更安全的疫苗形式也在不断涌现。近年来，一种具有“自主复制”功能的、以RNA病毒复制元件为基础的可复制型DNA疫苗的出现是DNA疫苗发展历程中的一个重大突破。这种疫苗集合了传统DNA疫苗、RNA疫苗以及RNA复制子疫苗的优势：(1)稳定性好，便于生产、储存和运输；(2)具有自我复制能力，外源基因可以得到高效表达；(3)转录和翻译过程均在宿主细胞的细胞质内进行，大大降低了外源基因与宿主细胞基因组整合的概率，提高了安全性；(4)由于其高效的复制和翻译，占用了宿主细胞的大部分资源，最终会引起宿主细胞的凋亡而被机体清除，降低了机体的免疫耐受。综上所述，可复制型DNA疫苗在治疗性疫苗的开发中显示出了广阔的应用前景。
本研究的目的在于构建一种基于塞姆利基森林病毒(Sen1ijkj forest vjms，SFv)的新型可复制型抗肿瘤DNA疫苗并对其抑瘤活性及免疫学作用机制进行深入的探讨。
首先，我们将含有红色和绿色荧光蛋白报告基因的片段“DsRed．．IRES—EGFP”克隆入本实验室前期构建保存的、经过卡那霉素(kfdl]a)抗性改造的基于SFv的PSCA(命名为PSCK)载体，以验证其真核表达能力；然后将本室验证具有良好抗肿瘤活性的DNA疫苗pwL)(1—2PFcGB中的多靶点融合抗原基因片段2PFcGB克隆入PSCK载体，构建成新型可复制型抗肿瘤DNA疫苗PSCK一2PFcGB，将该重组质粒瞬时转染293T细胞检测其在体外培养的细胞中的表达能力；通过肌肉注射加上电咏冲刺激的方法免疫小鼠，剥取免疫部位肌肉组织进行免疫组化分忻，对其活体内抗原的表达进行验证。
接着，我们采用PCR方法分别扩增出人Survivin、hCGB全长基因的cDNA编码区序列，利用DNA重组技术将其定向插入到真核表达载体pIRES一1]co中，利用阳离子脂质体转染和G418筛选技术，分别筛选出能够稳定表达人SuTvivin、hCGB的小鼠黑色素瘤(B16)细胞株；利用上述细胞株以不同浓度分别接种C57BL/6小鼠，观察稳定转染外源基因后B16细胞的致瘤能力，建立人survivin、hcGBI的黑色素瘤荷瘤小鼠模型。
随后，我们将编码荧光素酶基因的pGL3一CMV质粒利用肌肉注射、基因枪、电咏冲三种方式递送至BALB／c小鼠体内，在质粒递送后24—144h，连续检测小鼠体内的荧光素酶活性，通过荧光素酶活性的变化来比较经不同途径递送质粒DNA至BALB／c小鼠体内所诱导的表达效率。
最后，在上述研究结果的基础上，我们通过两种不同的免疫接种策略(治疗模型、予页防模型)来研究新型可复制型抗肿瘤DNA疫苗PSCK_2PFcGB的抗肿瘤效应。同时，从细胞免疫和体液免疫两个方面，对该疫苗所诱导的免疫学效应机制进行深入探讨。我们通过肌肉注射加电咏冲刺激的方式，将该疫苗递送至荷瘤C57BL／6小鼠体内，通过观察记录各组小鼠肿瘤生长情况来研究可复制型DNA疫苗的抑瘤活性；ElJISA法检测免疫小鼠血清中特异性抗体的滴度；LDH法体外检测免疫小鼠细胞毒性T淋巴细胞(CTL)反应；Elispot法检测免疫小鼠脾淋巴细胞IFN—v的分泌；流式细胞术和病理组织切片观察的方法检测免疫小鼠的离体肿瘤组织内的肿瘤浸润淋巴细胞。
结果证实：(1)经过kfdl]a抗性改造后的PS(：K载体能够高水平的表达红色和绿色荧光蛋白；成功构建了可复制型抗肿瘤DNA疫苗PS(：K_2PFcGB，利用流式细胞术及免疫组化法对该疫苗的体内外表达进行了验证。结果表明，IRES序列上游的融合抗原片段及下游的佐剂分子片段均获得良好的表达。(2)分别筛选出了能够稳定表达几SuTvivin、hCGB基因的B16细胞株，利用流式细胞仪、Weste：m blot、R_r_PCR及免疫荧光法检测了抗原分子的表达，为研究新型抗肿瘤DNA疫苗PSCK_2PFcGB的抑瘤作用机制提供了细胞模型；同时我们还建立了SuTvivhlI、hCGB’肿瘤细胞的C57BL／6荷瘤小鼠模型，为研究该疫苗的抑瘤活性研究提供了动物模型。(3)与传统的裸DNA质粒直接肌肉注射比较，电咏冲和基因枪均能有效的导入外源基因并提高外源基因在活体内的表达效率。相比较而言，电咏冲技术在肌肉注射DNA质粒后，加上电咏冲刺激即可完成，此方法可直接递送裸DNA质粒，操作简便、递送效率高、DNA的制备成本低廉。此外，DNA的储存和运输亦更加方便。(4)通过对免疫小鼠移植瘤生长趋势的观察和免疫学机制的研究，证明了新型可复制型抗肿瘤DNA疫苗PSCK_2PFcGB在两种免疫策略下均可有效地抑制肿瘤生长，并能诱导出较高水平的特异性细胞免疫和体液免疫应答。
本文主要以提高抗肿瘤DNA疫苗的免疫效力及增强疫苗安全性作为研究对象，在疫苗构建策略上，我们应用了基于SFV的可复制型DNA疫苗载体PSCK，该载体可以最大限度的提高外源基因的表达量且相对安全；在抗原选择上，将人Survivin的主要T细胞抗原表位区域和人、猴的hCGB—CTP37基因利用连接臂进行连接，构成异种化复合抗原2PAG，同时引入了人IgG Fc、GM—CSF和B7．1等免疫黏附分子、细胞因子及共刺激分子以增强免疫效力，含有该融合抗原片段的DNA疫苗pwL)(1—2PFcGB在动物实验中取得了较好的抗肿瘤效应。在靶细胞选择上，为了更好的评价疫苗抑瘤效果，我们建立了能稳定表达人Survivin、hCGB基因的小鼠黑色素瘤细胞模型及SurvivilI、hCGB’肿瘤细胞的荷瘤小鼠模型；在DNA疫苗递送方式上，我们对常用递送方式的递送效率进行了比较，选择了一种高效、稳定、便捷的疫苗递送途径；在免疫策略上，根据DNA疫苗的特性设计了两种不同的免疫方案，以此全面评估DNA疫苗的抑瘤效果。
综上所述，本研究在疫苗载体的应用、DNA递送方式和免疫策略的制定等多方面进行了新的尝试，证实所构建的新型可复制型抗肿瘤DNA疫苗PSCK一2PFcGB可有效地诱导免疫小鼠体内特异性的细胞免疫及体液免疫反应，能够有效地抑制移植瘤生长。这些结果为该疫苗进一步的药效学及药理学实验研究提供了依据、为该疫苗将来可能的临床应用奠定了良好的研究基础、为恶性肿瘤的予页防和治疗提供了新的方法和思路。
With the development of theory and technology in the field of Immunology,Molecular biology and Cell biology,the more efficient and more safer forms ofvaccines are emerging．In recent years，a new type of vector system based on RNAvirus replication components and with the fi．mction of”self-replication”has alreadybeen developed．It combines the advantages of traditional DNA vaccines，RNAvaccines and RNA replicon vaccines：(1)The replicon DNA vaccine has betterstability and ease of production,storage and transportation．(2)The presence of thereplicase genes leads to more mRNA replication resuking in the superior geneexpression．(3)The self-replication and transcription of the replicon DNA vaccineOCCULT in the cytoplasm,which eventually prevents the risk ofintegration into the hostcell genome and greatly improves the safety．(4)With efficient replication andtranslation,most ofthe resources ofthe host cell is consumed,eventually causing theapoptosis ofthe transfected cells，which will be cleared by the bodG and reducing theimmune tolerance．In summary,the replicon DNA vaccine will be expected to bepromising in the development oftherapeutic vaccines．
In order to develop a novel anti—tumor therapeutic vaccines，we constructed therecombinant based on the SFV virus and studied the antitumor efficacy and immunemechanism in vitro and in vivo．
In part I of this article，we inserted the gene fragments encoding red and greenfluorescent proteins into the PSCK vector and tested its expression in eukaryotic cells．Then,we inserted the mukitarget complex antigen 2PAG fusion gene(containing thecytotoxic T Lymphocyte epitopes of human Survivin and chorionic gonadotropin pchain—CTP37 of human and monky,human GM—CSF and B7．1，human IgG Fc andGPI anchor signal peptide)into the PSCK vector．Finally,we used flow cytometry andimmunohistochemistry to detect the expression offusion antigen．
In part II of this article，the full length of human Surviwin and hCGB cDNAfragments with the restriction enzyme position and 6×his tag were amplified by PCRand inserted into eukaryotic expression vector pIRES—neo．After identification of restriction digestion and PC R_The recombinant plasmids pIRES—neo—SUR-(his)6 andpIRES—neo_hCGB一(his)6 were obtained．Then the vectors were tramsfected into B16cells by lipofectamine 2000．After screening culture by G41 8，a stably tramsfected cellline was established,the transcription and expression of the human Survivin andhCGF gene was identified by RT-PC R_Western blot and immunofluorescence assay．Then,we used these two cell lines inoculated with C57BL／6 mouse in differentconcentration to established Survivin+，hCGF’melanoma tumor—bearing mice model．
Part III，BALB/C mice were administered with recombinant luciferase expressingplasmid pGL3--CMV or its mock plasmid：pGL3--basic via intramuscular injection,electric pulse and gene gun respectivey 10pg or 100~tg of the plasmids above wereadminstered via f m，or via electric pulse into musculus quadriceps fexoris．Theplasmids were intradermally deliverred by way of gene gun through 3 bullets，2pg ofplasmids and 4．5Mpa for each shoot．The luciferase activities were monitored over aperiod of 24—144 hour after gene delivery into mice by bioluminescence imaging invivo
Based on the previously study above，we developed two different strategies ofimmunization(treatment model and preventive model)to studied anti—tumor efficacyofthe replicative anti—tumor DNA vaccine PSCK-2PFcGB．At the same time，we alsostudied the mechanism of vaccine—induced cellular and humoral immunity．First，wedelivered the DNA vaccine into the tumor-bearing C57BL／6 mice musculusquadriceps fexoris via electric pulse，observed and recorded the tumor growth．Second，we used ELISA assay to detect the specific antibody titers in serum of immunizedmice；used LDH assay to discover the spleen lymphocytes specific CTL responses invitro；using immunized mice’S spleen to detect specific secreted IFN7 by Elispot assay．Finally_tumor infiltrating lymphocytes(TIL)in tumor tissues identified by flowcytometry and histopathology stain(H&E)assay in vitro．
The results confirmed that：(1)PSCK vector could express red and greenfluorescent protein in vitro，after identification of restriction digestion and PC R_thenovel replicative anti—tumor DNA vaccines PSCK-2PFcGFB was obtained，theexpression of fusion antigen gene and molecular adjuvant was well．(2)Theeukaryotic expression vector plRES--neo--hCGl3--(his)6 and plRES--neo--hCGl3--(his)6was successfully constructed．A stably tramsfected cell line was established and theexpression rate of Survivin,hCGl3 gene was higher than 90％respectively．Theexpression of the target gene provided a solid experimental foundation for further studies on tumor immunotherapy．Meanwhile，we also established a model ofSurviviI，hCGF tumor cells in C57BL／6 mice．(3)The desired high expression ofexogenous gene could be obtained by electric pulse and gene gun,and the expressionlevels via these two routes are much higher than that via f m．These methods arereliable methods for gene delivery~But，the electric pulse is simpler,higher efficiency,lower cost，the storage and transpo~is more convenient．(4)The results ofmorphological and study ofmechanism immunological showed the high antibody titerwas induced，the antigen specific IFN-r release was detected in immunized mice too，and it was detected that a strong correlation of CTL activity with protective efficiencyagainst tumor cells，such as inhibition of tumor growth，long lifespan and SO on inboth two different strategies ofimmunization(treatment model,preventive model)．
In this paper,in order to enhance the DNA vaccine efficacy and safety,we did alot ofworks．First，we applied the SFV-based DNA vaccine vector PSCK，this carriercan maximize the expression of foreign genes and relatively safe．About the tumorantigen,we used the fusion antigen fragment 2PFcGB contained the main T cellepitopes of Survivin and human,monkey hCGF—CTP37 genes(2PAG)，at the sametime，we also used some immune adhesion molecules，cytokines and costimulatorymolecules to enhance the immune effect such as human IgG Fc，GM—CSF and B7．1．In the choice of the target cell,we have established two stable expression of humanSurvivin,hCGp mouse melanoma(B16)model respectively and Survivin+，hCGp’tumor cells in mice．At the same time，we used bioluminescence technology to chooseone efficient，stable，convenient way to deliver the DNA vaccine．Finally,we havedesigned two different immunization strategies to evaluate the inhibitory effect ofDNA vaccines．
In summary,in this study,we had made a lot ofattemption in the application ofvaccine vector,DNA immunization strategies and delivery methods．The recombinantplasmid PSCK-2PFcGB was correctly constructed．The specific cellular and hnmoralimmune responses can be induced in higher levels after immunization with this novelkind ofreplicative anti-tumor DNA vaccine．These resuks provided a basis for furtherstudy in vaccine phaxmacodynamics and pharmacology and lay a solid foundation forclinical application．This kind ofhovel replicative anti—tumor DNA vaccines providesa new method and ideas for the prevention and treatment ofmalignant tumors．
本研究的目的在于构建一种基于塞姆利基森林病毒(Sen1ijkj forest vjms，SFv)的新型可复制型抗肿瘤DNA疫苗并对其抑瘤活性及免疫学作用机制进行深入的探讨。
首先，我们将含有红色和绿色荧光蛋白报告基因的片段“DsRed．．IRES—EGFP”克隆入本实验室前期构建保存的、经过卡那霉素(kfdl]a)抗性改造的基于SFv的PSCA(命名为PSCK)载体，以验证其真核表达能力；然后将本室验证具有良好抗肿瘤活性的DNA疫苗pwL)(1—2PFcGB中的多靶点融合抗原基因片段2PFcGB克隆入PSCK载体，构建成新型可复制型抗肿瘤DNA疫苗PSCK一2PFcGB，将该重组质粒瞬时转染293T细胞检测其在体外培养的细胞中的表达能力；通过肌肉注射加上电咏冲刺激的方法免疫小鼠，剥取免疫部位肌肉组织进行免疫组化分忻，对其活体内抗原的表达进行验证。
接着，我们采用PCR方法分别扩增出人Survivin、hCGB全长基因的cDNA编码区序列，利用DNA重组技术将其定向插入到真核表达载体pIRES一1]co中，利用阳离子脂质体转染和G418筛选技术，分别筛选出能够稳定表达人SuTvivin、hCGB的小鼠黑色素瘤(B16)细胞株；利用上述细胞株以不同浓度分别接种C57BL/6小鼠，观察稳定转染外源基因后B16细胞的致瘤能力，建立人survivin、hcGBI的黑色素瘤荷瘤小鼠模型。
随后，我们将编码荧光素酶基因的pGL3一CMV质粒利用肌肉注射、基因枪、电咏冲三种方式递送至BALB／c小鼠体内，在质粒递送后24—144h，连续检测小鼠体内的荧光素酶活性，通过荧光素酶活性的变化来比较经不同途径递送质粒DNA至BALB／c小鼠体内所诱导的表达效率。
最后，在上述研究结果的基础上，我们通过两种不同的免疫接种策略(治疗模型、予页防模型)来研究新型可复制型抗肿瘤DNA疫苗PSCK_2PFcGB的抗肿瘤效应。同时，从细胞免疫和体液免疫两个方面，对该疫苗所诱导的免疫学效应机制进行深入探讨。我们通过肌肉注射加电咏冲刺激的方式，将该疫苗递送至荷瘤C57BL／6小鼠体内，通过观察记录各组小鼠肿瘤生长情况来研究可复制型DNA疫苗的抑瘤活性；ElJISA法检测免疫小鼠血清中特异性抗体的滴度；LDH法体外检测免疫小鼠细胞毒性T淋巴细胞(CTL)反应；Elispot法检测免疫小鼠脾淋巴细胞IFN—v的分泌；流式细胞术和病理组织切片观察的方法检测免疫小鼠的离体肿瘤组织内的肿瘤浸润淋巴细胞。
结果证实：(1)经过kfdl]a抗性改造后的PS(：K载体能够高水平的表达红色和绿色荧光蛋白；成功构建了可复制型抗肿瘤DNA疫苗PS(：K_2PFcGB，利用流式细胞术及免疫组化法对该疫苗的体内外表达进行了验证。结果表明，IRES序列上游的融合抗原片段及下游的佐剂分子片段均获得良好的表达。(2)分别筛选出了能够稳定表达几SuTvivin、hCGB基因的B16细胞株，利用流式细胞仪、Weste：m blot、R_r_PCR及免疫荧光法检测了抗原分子的表达，为研究新型抗肿瘤DNA疫苗PSCK_2PFcGB的抑瘤作用机制提供了细胞模型；同时我们还建立了SuTvivhlI、hCGB’肿瘤细胞的C57BL／6荷瘤小鼠模型，为研究该疫苗的抑瘤活性研究提供了动物模型。(3)与传统的裸DNA质粒直接肌肉注射比较，电咏冲和基因枪均能有效的导入外源基因并提高外源基因在活体内的表达效率。相比较而言，电咏冲技术在肌肉注射DNA质粒后，加上电咏冲刺激即可完成，此方法可直接递送裸DNA质粒，操作简便、递送效率高、DNA的制备成本低廉。此外，DNA的储存和运输亦更加方便。(4)通过对免疫小鼠移植瘤生长趋势的观察和免疫学机制的研究，证明了新型可复制型抗肿瘤DNA疫苗PSCK_2PFcGB在两种免疫策略下均可有效地抑制肿瘤生长，并能诱导出较高水平的特异性细胞免疫和体液免疫应答。
本文主要以提高抗肿瘤DNA疫苗的免疫效力及增强疫苗安全性作为研究对象，在疫苗构建策略上，我们应用了基于SFV的可复制型DNA疫苗载体PSCK，该载体可以最大限度的提高外源基因的表达量且相对安全；在抗原选择上，将人Survivin的主要T细胞抗原表位区域和人、猴的hCGB—CTP37基因利用连接臂进行连接，构成异种化复合抗原2PAG，同时引入了人IgG Fc、GM—CSF和B7．1等免疫黏附分子、细胞因子及共刺激分子以增强免疫效力，含有该融合抗原片段的DNA疫苗pwL)(1—2PFcGB在动物实验中取得了较好的抗肿瘤效应。在靶细胞选择上，为了更好的评价疫苗抑瘤效果，我们建立了能稳定表达人Survivin、hCGB基因的小鼠黑色素瘤细胞模型及SurvivilI、hCGB’肿瘤细胞的荷瘤小鼠模型；在DNA疫苗递送方式上，我们对常用递送方式的递送效率进行了比较，选择了一种高效、稳定、便捷的疫苗递送途径；在免疫策略上，根据DNA疫苗的特性设计了两种不同的免疫方案，以此全面评估DNA疫苗的抑瘤效果。
综上所述，本研究在疫苗载体的应用、DNA递送方式和免疫策略的制定等多方面进行了新的尝试，证实所构建的新型可复制型抗肿瘤DNA疫苗PSCK一2PFcGB可有效地诱导免疫小鼠体内特异性的细胞免疫及体液免疫反应，能够有效地抑制移植瘤生长。这些结果为该疫苗进一步的药效学及药理学实验研究提供了依据、为该疫苗将来可能的临床应用奠定了良好的研究基础、为恶性肿瘤的予页防和治疗提供了新的方法和思路。
With the development of theory and technology in the field of Immunology,Molecular biology and Cell biology,the more efficient and more safer forms ofvaccines are emerging．In recent years，a new type of vector system based on RNAvirus replication components and with the fi．mction of”self-replication”has alreadybeen developed．It combines the advantages of traditional DNA vaccines，RNAvaccines and RNA replicon vaccines：(1)The replicon DNA vaccine has betterstability and ease of production,storage and transportation．(2)The presence of thereplicase genes leads to more mRNA replication resuking in the superior geneexpression．(3)The self-replication and transcription of the replicon DNA vaccineOCCULT in the cytoplasm,which eventually prevents the risk ofintegration into the hostcell genome and greatly improves the safety．(4)With efficient replication andtranslation,most ofthe resources ofthe host cell is consumed,eventually causing theapoptosis ofthe transfected cells，which will be cleared by the bodG and reducing theimmune tolerance．In summary,the replicon DNA vaccine will be expected to bepromising in the development oftherapeutic vaccines．
In order to develop a novel anti—tumor therapeutic vaccines，we constructed therecombinant based on the SFV virus and studied the antitumor efficacy and immunemechanism in vitro and in vivo．
In part I of this article，we inserted the gene fragments encoding red and greenfluorescent proteins into the PSCK vector and tested its expression in eukaryotic cells．Then,we inserted the mukitarget complex antigen 2PAG fusion gene(containing thecytotoxic T Lymphocyte epitopes of human Survivin and chorionic gonadotropin pchain—CTP37 of human and monky,human GM—CSF and B7．1，human IgG Fc andGPI anchor signal peptide)into the PSCK vector．Finally,we used flow cytometry andimmunohistochemistry to detect the expression offusion antigen．
In part II of this article，the full length of human Surviwin and hCGB cDNAfragments with the restriction enzyme position and 6×his tag were amplified by PCRand inserted into eukaryotic expression vector pIRES—neo．After identification of restriction digestion and PC R_The recombinant plasmids pIRES—neo—SUR-(his)6 andpIRES—neo_hCGB一(his)6 were obtained．Then the vectors were tramsfected into B16cells by lipofectamine 2000．After screening culture by G41 8，a stably tramsfected cellline was established,the transcription and expression of the human Survivin andhCGF gene was identified by RT-PC R_Western blot and immunofluorescence assay．Then,we used these two cell lines inoculated with C57BL／6 mouse in differentconcentration to established Survivin+，hCGF’melanoma tumor—bearing mice model．
Part III，BALB/C mice were administered with recombinant luciferase expressingplasmid pGL3--CMV or its mock plasmid：pGL3--basic via intramuscular injection,electric pulse and gene gun respectivey 10pg or 100~tg of the plasmids above wereadminstered via f m，or via electric pulse into musculus quadriceps fexoris．Theplasmids were intradermally deliverred by way of gene gun through 3 bullets，2pg ofplasmids and 4．5Mpa for each shoot．The luciferase activities were monitored over aperiod of 24—144 hour after gene delivery into mice by bioluminescence imaging invivo
Based on the previously study above，we developed two different strategies ofimmunization(treatment model and preventive model)to studied anti—tumor efficacyofthe replicative anti—tumor DNA vaccine PSCK-2PFcGB．At the same time，we alsostudied the mechanism of vaccine—induced cellular and humoral immunity．First，wedelivered the DNA vaccine into the tumor-bearing C57BL／6 mice musculusquadriceps fexoris via electric pulse，observed and recorded the tumor growth．Second，we used ELISA assay to detect the specific antibody titers in serum of immunizedmice；used LDH assay to discover the spleen lymphocytes specific CTL responses invitro；using immunized mice’S spleen to detect specific secreted IFN7 by Elispot assay．Finally_tumor infiltrating lymphocytes(TIL)in tumor tissues identified by flowcytometry and histopathology stain(H&E)assay in vitro．
The results confirmed that：(1)PSCK vector could express red and greenfluorescent protein in vitro，after identification of restriction digestion and PC R_thenovel replicative anti—tumor DNA vaccines PSCK-2PFcGFB was obtained，theexpression of fusion antigen gene and molecular adjuvant was well．(2)Theeukaryotic expression vector plRES--neo--hCGl3--(his)6 and plRES--neo--hCGl3--(his)6was successfully constructed．A stably tramsfected cell line was established and theexpression rate of Survivin,hCGl3 gene was higher than 90％respectively．Theexpression of the target gene provided a solid experimental foundation for further studies on tumor immunotherapy．Meanwhile，we also established a model ofSurviviI，hCGF tumor cells in C57BL／6 mice．(3)The desired high expression ofexogenous gene could be obtained by electric pulse and gene gun,and the expressionlevels via these two routes are much higher than that via f m．These methods arereliable methods for gene delivery~But，the electric pulse is simpler,higher efficiency,lower cost，the storage and transpo~is more convenient．(4)The results ofmorphological and study ofmechanism immunological showed the high antibody titerwas induced，the antigen specific IFN-r release was detected in immunized mice too，and it was detected that a strong correlation of CTL activity with protective efficiencyagainst tumor cells，such as inhibition of tumor growth，long lifespan and SO on inboth two different strategies ofimmunization(treatment model,preventive model)．
In this paper,in order to enhance the DNA vaccine efficacy and safety,we did alot ofworks．First，we applied the SFV-based DNA vaccine vector PSCK，this carriercan maximize the expression of foreign genes and relatively safe．About the tumorantigen,we used the fusion antigen fragment 2PFcGB contained the main T cellepitopes of Survivin and human,monkey hCGF—CTP37 genes(2PAG)，at the sametime，we also used some immune adhesion molecules，cytokines and costimulatorymolecules to enhance the immune effect such as human IgG Fc，GM—CSF and B7．1．In the choice of the target cell,we have established two stable expression of humanSurvivin,hCGp mouse melanoma(B16)model respectively and Survivin+，hCGp’tumor cells in mice．At the same time，we used bioluminescence technology to chooseone efficient，stable，convenient way to deliver the DNA vaccine．Finally,we havedesigned two different immunization strategies to evaluate the inhibitory effect ofDNA vaccines．
In summary,in this study,we had made a lot ofattemption in the application ofvaccine vector,DNA immunization strategies and delivery methods．The recombinantplasmid PSCK-2PFcGB was correctly constructed．The specific cellular and hnmoralimmune responses can be induced in higher levels after immunization with this novelkind ofreplicative anti-tumor DNA vaccine．These resuks provided a basis for furtherstudy in vaccine phaxmacodynamics and pharmacology and lay a solid foundation forclinical application．This kind ofhovel replicative anti—tumor DNA vaccines providesa new method and ideas for the prevention and treatment ofmalignant tumors．
引文
[1]汪楠，田玲，王敏．癌症疫苗发展现状与趋势分忻[J]l中国疫苗和免疫．2010，16f4)：371-374．
[2]高云华，吴曙霞，刁天喜．癌症疫苗产业市场的现状与发展趋势[J]．中国新药杂志．2008，17(19)：1641—1643．
[3]McArdle SE，Rees RE．Tumor immunology：new perspectives[J]l Cancer Res，2009，69f18)：7157—7159．
[4]Kenneth Lundstrom．Alphaviruses in Gene Therapy[J]lViruses，2009，1《1)，13—25．
[5]Schlesinger S．A~havirus vectors：development mad potential therapeuticapplications．Expert Opin Biol Ther，2001，1(2)：177—191．
[6]Jonathan O R，Sergey AD，Kurt IK．A~havirus vectors and vaccination[J]l RevMed Virol,2002，12：279—296．
[7]余云舟，孙志伟，刘志刚等．基于DNA的SemlNi森林病毒复制子载体体内外高水平表达外源基因[J]l生物化学与生物物理进展．Progress in Biochemistryand Biophysics，2006，33(1)：87—94．
[8]Sun Y，D Liu，et aL A prime_boost vaccination~rNegy using a Seml~i Forestvirus replicon ve~ored DNA vaccine followed by a recombinant adenovirus protectspigs from classical swine fever[J]l Sheng Wu Gong Cheng Xue Bao．2009，25《5)：679—685．
[9]Quintaxm—Vazquez D，Vazquez—Blomquist DM，Galban Rodriguez E，et aL Avaccination strategy consisting of Semliki—Forest—virus fSFVl DNA prime andfoWlDOX—virus boost significantl,／DrotectS mice from a recombinant rHIV一1)-vaccinia-virus infection[J]l Biotechnol Appl Biochem，2005，41《1)：59-66．
[10]Durso R．J，Andjelic S，Gardner J．E et a1．Novel alphavirus vaccine encodingprostate-specific membrane antigen elicits potent cellular and humoral immuneresponses．Clin．Cancer Res．2007，13，3999-4008．[1 1]Gupta P K，SS Dahiya,et al Sindbis virus replicon-based DNA vaccine encodingRabies virus glycoprotein elicits specific humoral and cellular immune response indogs[J]l Acta Vir01．2009，53(2)：83-88．
[12]Riezebos-Brilman A_Regts J，Chen M，et al Augmentaion ofalphavirusvector--induced human papilloma virus--specific immune and anti--tumour responses bycoexpression ofinterleukin-12．Vaccine 2009，27，701-707．
[13]Gabler M，Scheiblhofer S，Kern K，et a1．Immunization with a low-dose repliconDNA vaccine encoding Phl P 5 effectively prevents allergic sensitization[J]．JAllergy Clin Immunol,2006，1 18(3)：734-41．
[14]David I．Bernsteina，Elizabeth A．Reapb，Kevin Katenb,et a1．Randomized,double-blind，Phase 1 trial ofan alphavirus replicon vaccine for cytomegalovirus inCMV seronegative adult volunteers[J]l Vaccine，20 10，28：484-493．
[15]罗颖．Survivin多肽疫苗治疗血液系统恶性肿瘤的研究进展[J]．重庆医科大学学报，2007，32(10)：1118-1120．
[16]杜冠辉，谭金风，吴淑金，李强Survivin与肿瘤的关系[J]l医学综述，2008，14 f51：658-660．
[17]Marioni G，ED’Alessandro，et aL Survivin multifaceted activity in head and neckcarcinoma：current evidence and future therapeutic challenges[J]．Acta Otolaxyng01．2010，130(1)：4-9．
[18]Tetsuhiro T，Yuji I，Tosei，et aL Clinical and immunological evaluation ofanti-apoptosis protein,survivin-derived peptide vaccine in phase I clinical study forpatients with advanced or recurrent breast cancer[J]l J Transl Med．2008，6(24)：1-1 1．
[19]Miyazaki A_J Kobayashi，et aL Phase I clinical trial of survivin-derived peptidevaccine therapy for patients with advanced or recurrent oral cancer[J]l Cancer Sci201 1，102(2)：324-329．
[20]Cole LA．Biological functions of hCG and hCG-related molecules[J]．ReprodBiol Endocrino．2010．1 8：102．
[21]李明清，唐传玲．抗hCG疫苗在肿瘤免疫治疗中的价值[J]．中国免疫学杂志，2008，24(12)：1149-1151．
[22]Qing Dong Guan,Ying Wang，Yi Wei Chu，chu．The distinct effects of threetandem repeats of C3d in the immune responses against tumor-associated antigenhCG by DNA immunization[J]l Cancer Immunol Immunother．2007，56：875-884．
[23]Xiangbing，H．，Z．Yankai，el a1．The fusion protein of HSP65 with tandemrepeats ofbeta-hCG acting as a potent tumor vaccine in suppressing hepatocarcinoma[J]l Int Immunopharmac01．2010，10(2)：230-238．
[24]Gupta R，Emens LA．GM-CSF-secreting vaccines for solid tumors：movingforward[J]．Discov Med．20 10．10(50)：52-60．
[25]Burke JM．GM-CSF-alTned replication-competent viruses for cancer[J]lCytokine Growth Factor Rev．2010．21(2-3、：149-151
[26]Clive KS，Tyler JA，Clifton GL el al Use of GM-CSF as an adjuvant withcancer vaccines：beneficial or detrimental[J]?Expert Rev Vaccines．20 10，9(5)：5 19-525．
[27]Arnett HA，JL Viney．Recent developments in IBD and the B7 family ofcostimulatory molecules[J]l Curr Opin Investig Drugs．2009，10(5)：443-45 1．
[28]Petroff MG A Perchellet．B7 fanljly molecules as regulators of the maternalimmune system in pregnancy[J]l Am J Reprod Immun01．2010，63(6)：506-519．
[29]Loos M，DM Hedderich，el a1．B7-h3 and its role in antitumor immunity[J]l ClinDev Immun01．2010，683875．
[30]Webster ML，G Zhu，el a1．Fc-independent phagocytosis；implications forintravenous IgG therapy in immune thrombocytopenia[J]．Cardiovasc HematolDisord Drug Targets．2008，8(4)：278-282．[3 1]Anthony RM，JV Ravetch．A novel role for the IgG Fc glTy’can；theanti-inflammatory activity of sialylated IgG Fcs[J]l J Clin Immun01．2010，30 Suppl 1：S9-14．
[32]Lejeune J，G Thibault，el a1．Implications of receptors for the Fc portion of IgG《FcgammaRs)in mechanism of action oftherapeutic antibodies[J]l Bull Cancer．20 10，97f51：511-522．
[33]Sesarman A，G Vidarsson,el a1)．The neonatal Fc receptor as therapeutic targetin IgG-mediated autoimmune diseases[J]l Cell Mol Life Sci．2010，67(15)：2533-2550．
[34]Ferrone C，Perales M，Goldberg S，el aL Adjuvanticity ofplasmid DNA encodingcytokines fused to immunoglobulin Fc domains[J]．Clin Cancer Res，2006，12(18)：5511-5519．
[35]Sh oji Hosaka E，Kobayashi Y，Wakitani M，el a1．Enhanced Fc dependent cellularcytotoxicity of Fc fusion proteins derived from TNF receptor II and LFA-3 by fucoseremoval fromAsh linked oligosaccharides[J]．J Biochem,2006，140《6)：777-783．
[36]FujjIa M，Y Jigami．Lipid remodeling of GPI-anchored proteins and its function[J]．Biochim Biophys Acta．2008，1780(3)：410-420．
[37]Fujita M，T Kinoshita．Structural remodeling ofGPI anchors during biosynthesisand after attachment to proteins[J]l FEBS Lett．2010，584(9)：1670-1677．
[38]Heller LC，Ugen K，Heller R．Electroporation for targeted gene transfer[J]l Exp．Opin．Drug Deliv,2005，2(2)：255-268．
[39]Luckay A_Sidhu M K，Kjeken R，et aL Effect of plasmid DNA vaccine desingand in vivo electroporation on the resulting vaccine-specific immune responses inrhesus macaques[J]l J．Virol,2007，81《10)：5257-5269．
[40]Hooper J，Golden J，FeHo A，et aL Smallr}ox DNA vaccine delivered by novelskin electroporation device protects mice against intranasal poxvirus challenge[J]．Vaccine，2007，25(10)：1814-1823．
[41]Neumann E，Schacfer-Ridder M，Wang，et al Gene transfer into mouse lyomacells by electroporation in high electric fields[J]．EMBO J，1982，《1)：841-845．
[1]Kenneth Lundstrom．Alphaviruses in Gene Therapy[J]lViruses，2009，1(1)，13—25．
[2]王红仁，李明远．基于甲病毒的RNA复制子疫苗[J]l微生物学免疫学进展，2007，35f2)：72—75．
[3]余云舟，孙志伟，刘志刚等．基于DNA的Semliki森林病毒复制子载体体内外高水平表达外源基因[J]．生物化学与生物物理学进展，2006，33(1)：87—94．
[4]王昌青，杨兵．DNA疫苗研究进展[J]l中国畜牧兽医，2009，36(12)：143—147．
[5]Ledwith BJ，Manam S，Troilo PJ，et a1．Plasmid DNA vaccines：assay forintegration into host genomic DNA[J]l Dev Biol(Basel)，2000，104：33—43．
[6]Wang Z，TroiloP J，Wang X，el a1．Detection of integration of plasmid DNA intohost genomic DNA following intramuscular injection and electroporation[J]．GeneTher,2004，11(8)：711-721．
[7]Gupta P K，SS Dahiya，el al Sindbis virus replicon-based DNA vaccine encodingRabies virus glycoprotein elicits specific humoral and cellular immune response indogs[J]l Acta Virok 2009，53(2)：83—88．
[8]Sun Y，D Liu，el a1．A prime—boost vaccination strategy using a Semliki Forestvirus replicon vectored DNA vaccine followed by a recombinant adenovirus protectspigs from classical swine fever[J]l Sheng Wu Gong Cheng Xue Bao．2009，25(5)：679—685．
[9]Riezebos—BrilmanA，RegtS J，Chen M，el a1．Augmentation ofall)havirusvector--induced human papilloma virus--specific immune and anti--tumour responses bycoexpression ofinterleukin-12．Vaccine 2009，27，701-707．
[10]M／i~itt／i A．M，MSJcinen K，Ketola A_el a1．Replication competent SemlNi Forestvirus prolongs survival in experimental lung cancer．Int．J．Cancer 2008，123，1704—171 1．
[11]Quinn,K．Effect ofintranasal administration ofSemliki Forest virus recombinantparticles expressing interferon-[3 on the progression of experimental autoimmuneencephalomyelitis．M01．Med．Rep．2008，1，335—342．
[12]Zheng M，N Jin,el a1．Immunogenicity and protective efficacy of Semliki forestvirus replicon-based DNA vaccines encoding goatpox virus structural proteins[J]lVirology．2009，391(1)：33—43．
[13]Lundstrom,K．Biology and application ofalphaviruses in gene therapy[J]l GeneTher．2005，Suppl．1，$92—97．
[14]Durso R．J，Andjelic S，Gardner J．P，el a1．Novel alphavirus vaccine encodingprostate—specific membrane antigen elicits potent cellular and humoral immuneresponses．Clin．Cancer Res．2007，13，3999—4008．
[15]Leitner w_Hwang L，Bergmann L，el a1．Apoptosis is essential for the increasedefficacy of alphaviral replicase—based DNA vaccines[J]l Vaccine，2004，22(1 1-12)：1537—1 544
[1]Ambrosini G，Adida C，Altieri D．el aL A novel an apoptosis gene，Survivin,expressed in cancer and lymphoma[J]．Nat Med，1997，3(8)：917—921．
[2]罗颖．Survivin多肽疫苗治疗血液系统恶性肿瘤的研究进展[J]l重庆医科大学学报，2007，32(10)：1118-1120．
[3]杜冠辉，谭金风，吴淑金，李强Survivin与肿瘤的关系[J]l医学综述，2008，14 (5)：658—660．
[4]Marioni G，ED’Alessandro，el aL Survivin multifaceted a~ivity in head and neckcarcinoma：current evidence and future therapeutic challenges[J]l Acta Otolaryng01．2010，130(1)：4—9．
[5]Ulf-Hakan S，Henrik A_Kristina H．Human chorionic gonadotropin in cancer[J]lClinical Biochemistry 2004，(37)：549—561．
[6]Cole LA．Biological fi．mctions of hCG and hCG—related molecules[J]l ReprodBiol Endocrino．2010．1 8：102．
[7]李明清，唐传玲．抗hCG疫苗在肿瘤免疫治疗中的价值[J]．中国免疫学杂志，2008，24(12)：1149—1151．
[8]王永芬，马辉，吴学军．抗人绒毛膜促性腺激素单克隆抗体的制备与鉴定[J]l细胞与分子免疫学杂志2008，24(1 1)：1 102—1 103．
[9]Tetsuhiro T，Yuji I，Tosei．el al Clinical and immunological evaluation ofanti-apoptosis protein,survivin—derived peptide vaccine in phase I clinical study forpatients with advanced or recurrent breast cancer[J]l J Tramsl Med．2008，6(24)：1-1 1．
[10]Yamg Z，L Wang，el aL A novel mimovirus vaccine containing survivin epitopewith adjuvant IL一15 induces long—lasting cellular immunity and high antitumorefficiency[J]．Mol Immun01．2008，45(6)：1674—1681．
[11]Li H，MH Li，el aL Human survivin modified DCs vaccine inhibits laryngealcancer in vivo and in vitro[J]．Zhonghua Er Bi Yah Hou Tou Jing Wai Ke Za Zhi2009，44f9)：767—771．
[12]Miyazaki A_J Kobayashi，et aL Phase I clinical trial of survivin-derived peptidevaccine therapy for patients with advanced or recurrent oral cancer[J]．Cancer Sci2011，102(2)：324—329．
[13]Xiangbing H，Z Yankai，et aL The fusion protein ofHSP65 with tandem repeatsof beta—hCG acting as a potent tumor vaccine in suppressing hepatocarcinoma[J]l IntImmunopharmacol 2010，10(2)：230—238．
[14]P．J．Delves，R．K．Iles，I．M．Roitt，et a1．Designing a new generation ofanti—hCGvaccines for cancer therapy[J]l Molecular and Ce~ular Endocrinology 2007(260-262)：276-281．
[15]He Yi，Yaxl Rong，Zhang Yankai，et aL Improved efficacy of DNA vaccinationagainst breast cancer by boosting with the repeat beta-hCG C—terminal peptide carriedby mycobacte heat—shock protein HSP65[J]．Vaccine 2006，24(14)：2575—2584．
[16]Rong X，Noriko M，Yunping L．et aL DNA Vaccine Targeting SurvivinCombines Apoptosis with Suppression of Angiogenesis in Lung Tumor Eradication[J]l Cancer Res 2005，65(2)：553—561．
[17]Guan QD，Wang Y，Chu Yw，eL The distinct evects ofthree tandem repeatsof C3d in the immune responses against tumor—associated antigen hCGB by DNAimmunization[J]．Cancer Immunol Immun 2007，56(6)：875—884．
[18]Kitamura H，Torigoe T，Honma I．et aL expression and antigenicity of survivin,an inhibitor of apoptosis family member，in bladder cancer：implications for specificimmunotherapy[J]．Urology，2006，67(5)：955—959．
[19]Abad JD，Wrzensinski C，Overwijk W，et aL T—cell receptor gene therapy ofestablishedtumorsin amurinemelanomamodel[J]l JImmunother，2008，31(1)：1-6．
[20]Guimaraes Ferreira CA，Rodrigues EG，Mortara RA，et aL Antitumor effects invitro and in vivo and mechanisms ofprotection against melanoma B16F10一Nexcellsby fastuosain，a cysteine proteinase fcom Bromelia fastuosa[J]l Neoplasia,2007，9(9)：723．733
[1]Koyama S，T Kawagoe．et aL Immunological mechanism ofDNA vaccine efficacy[J]l Tanpakushitsu Kakusan Koso 2009，54(8 Suppl)：1096—1100．
[2]Jana B，Milada H，Michal K．et aL DNA vaccines：are they still just a powerfultool for the future[J]?Arch．Immun01．Ther．Exp．2007，55，387—398
[3]lJu S，Wang S，Jill M，et aL Current progress of DNA vaccine studies in humans[J]l Expert Rev．Vaccine，2008，7(2)：175—191．
[4]Wu TC．Therapeutic human papillomavirus DNA vaccination strategies to controlcervical cancer[J]．Eur．J．Immun．2007，37(2)：310—314．
[5]Bedognetti D，E．Wang，et aL Gene—expression profiling in vaccine therapy andimmunotherapy for cancer[J]l Expert Rev Vaccines．2010，9(6)：555—565．
[6]Kieback，E，W．Uckert．Enhanced T cell receptor gene therapy for cancer[J]．Expert Opin Biol Ther．2010，10(5)：749—762．
[7]Samaranayake，H，A．M．Maatta，et aL Future prospects and challenges ofantiangiogenic cancer gene therapy[J]l Hum Gene Ther．2010，21(4)：381—396．
[8]Marhsall E．Gene therapy death prompts review of adenovirus vector[J]l Science，2000，286：2244—2245．
[9]Mammohan S，Aravind C，Derek OH，et aL Nanopaxticles and micropazticles asvaccine delivery systems[J]l Expert Rev．Vaccines，2007，6(5)：797—808．
[10]何保凌，孙恩杰，黄文．基因枪技术及其在基因治疗中的应用进展[J]l生物学杂志2007，24(2)：47—50．
[10]Fuller D，Loudon P，SchmaUohn C．Preclinical and clinical progress ofparticle—medi~ed DNA vaccines for infectious diseases[J]l Methods，2006，40(1)：86—97．
[11]Sharpe M，Lynch D，Topham S，et aL Protection of mice from H5N1 influenzachal~nge by prophylactic DNA vaccination using particle mediated epidermaldelivery[J]l Vaccine，2007，25(34)：6392—6398．
[12]Neumann E，Schacfer-Ridder M，Wang，et al Gene transfer into mouse lyomacells by electroporation in high electric fields[J]l EMBO J，1982，(1)：841—845．
[13]Ionescu—zanetti C，Blatz A_Khine M，et aL Electrophoresis—assisted single—cellelectroporation for efficient intracellulax delivery[J]．Biomed Micro devices，2008，10(1)：113—116．
[14]Luckay A_Sidhu M K，Kjeken R，et aL Effect of plasmid DNA vaccine desingand in vivo electroporation on the resuking vaccine—specific immune responses inrhesus macaques[J]l J．Virol,2007，81(10)：5257—5269．
[15]Hooper J，Golden J，FeHo A，et aL Smallr}OX DNA vaccine delivered by novelskin electroporation device protects mice against intranasal poxvkus chal~nge[J]lVaccine，2007，25(10)：1814—1823．
[16]Valero A_Post JN，Van Nieuwkasteele Jw et aL Gene transfer and proteindynamics in stem cells using single cell electroporation in amicrofluidic device[J]lLab Chip，2008，8(1)：62—67．
[1]DouganM，G Dranofl：Immunetherapy for cancer[J]l Annu RevImmun01．2009，27：83—117．
[2]Pellegrini，M，Tw Mak．Tumor immune therapy：lessons from infection andimplications for cancer—can IL-7 help overcome immune inhibitory networks[J]?Eur J Immun01．2010，40(7)：1852—1861．
[3]Ikeda H，N Imai，el al Cancer treatment with gene—modified T cells or DNAvaccine[J]l Nippon Rinsho 2010，68(Suppl 8)：634—640．
[4]Becker J．L BM Olson,el al DNA vaccine encoding prostatic acid phosphatase(PAP)elicits long—term T-cell responses in patients with recurrent prostate cancer[J]lJ Immuno ther．2010，33(6)：639—647．
[5]Sedgwick JD．ELISPOT assay：a personal retrospective．Methods Mol Biol,2005，302(1)：3—14．
[6]Wolff JA，Malone RW,Williams P，el al Direct gene transfer into mouse musclein vivo[J]l Science，1990，247(49)：1465—1468．
[7]Koyama S，T Kawagoe，el al Immunological mechanism of DNA vaccineefficacy[J]l Tanpakushitsu Kakusan Koso 2009，54(8 Supp~：1096—1100．
[8]Sham L，Shixiaw，JillM，el a1．Charrent progressofDNA vaccine studiesin humans[J]l Expert Rev．Vaccines,2008，7(2)：175—191．
[9]Jaxm B，Milada H，Michal K，el a1．DNA vaccines：are they stilljust a powerful toolforthefuture[J]?Arch．Immunol,Ther．Exp．2007，55：387-398．
[10]Xuezw，XLZhmt Research progress onDNAtulTlor vaccine[J]．Zhongguo YiMlao He Mlan Y1．2U10，16(2)：183—186．
[11]WuWC，DYjin,etal．Inductionofaxltigen-specificCTL and antibody responsesinmice by a novel recombinant tandem repeat DNA vaccine targeting at mucin 1 ofpancreatic cancer[J]．J Cancer Res．Clin Oncok 2010，136(12)：1861—1868．
[12]Chain RC，B．Gutierrez，el aL Enhancement ofDNA cancer vaccine efficacy bycombination with anti-angiogenesis in regression of estab~shed subcutaneous B16melanoma[J]．Oncol Rep 2009，22(5)：1 197—1203．
[13]Lai MD，MC Yen,el aL The effects of DNA formulation and administration routeon cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung canceranimal model[J]．Genet Vaccines Ther．2009，7：2．
[14]Pakravam N_L Lamgroudi，el aL Co—adminiaration of GP96 and Her2／neu DNAvaccine in a Her2 breast cancer model[J]．Cell Stress Chaperones．2010，15(6)：977—984．
[15]McNeel DG，EJ Dunphy，el aL Safety and immunological efficacy of a DNAvaccine encoding prostatic acid phosphatase in patients with stage DO prostate cancer[J]．J C1in Oncok 2009，27(25)：4047—4054．
[16]Becker JT，BM．Olsork el aL DNA vaccine encoding prostatic acid phosphatase(PAP)elicits long-term T—cell responses in patients with recurrent prostate cancer[J]．JImmunother-2010，33(6)：639—647．
[1]Kenneth Lundstrom．Alphaviruses in Gene Therapy[J]lViruses，2009，1(1)，13—25．
[2]Schlesinger S．A~havkus vectors：development and potential therapeuticapplications．Expeg Opin Biol Ther，2001，1(2)：177—191．
[3]Jonathan O R，Sergey AD，Kurt IK．A~havirus vectors and vaccination[J]l RevMed Virol,2002，12：279—296．
[4]David I．Bernsteina,Elizabeth A．Reapb，Kevin Katenb,el aL Randomized，double—blind，Phase 1 trial ofan alphavirus replicon vaccine for cytomegalovirus inCMV seronegative adult volunteers[J]l Vaccine，2010，28：484—493．
[5]Lundstrom,K．Biology and application of alphaviruses in gene therapy[J]l GeneTher．2005，Suppl．1，$92—97．
[6]Durso R．J，Andjelic S，Gardner J．P et a1．Novel alphavirus vaccine encodingprostate—specific membrane antigen elicits potent cellular and humoral immuneresponses．Clin．Cancer Res．2007，13，3999—4008．
[7]Gupta P K，SS Dahiya，et al Sindbis virus replicon-based DNA vaccine encodingRabies virus glycoprotein elicits specific humoral and cellular immune response indogs[J]．Acta Virol 2009，53(2)：83—88．
[8]Saxena S，SS Dabiya，el aL A sindbis virus replicon-based DNA vaccine encodingthe rabies virus glycoprotein elicits immune responses and complete protection inmice from~thal chal~nge[J]．Vaccine 2008，26(51)：6592—6601．
[9]Zheng M，N Jin,et aL Immunogenicity and protective efficacy of Semliki forestvirus replicon-based DNA vaccines encoding go．pox virus structural proteins[J]lVirology．2009，391(1)：33—43．
[10] Li N，YZ Yu，et aL Enhancement of the immunogenicity of DNA repliconvaccine of Clostridium botulinum neurotoxin serotype A by GM—CSF gene adjuvant[J]l Immunopharmacol Immunotoxic01．201 1，33(1)：21 1-219．
[11]Yu Y J Yu, eL Individual and bivalent vaccines against botulinum neurotoxinserotypes A and B using DNA-based Semliki Forest virus vectors[J]l Vaccine．2009，27(44)：6148—6153．
[12]Sun Y N Li，et aL Enhanced immunity against classical swine fever in pigsinduced by prime—boost immunization using an alphavirus replicon-vectored DNAvaccine and a recombinant adenovirus[J]l Vet Immunol Immunopath01．2010，137(1—21：20—27．
[2]高云华，吴曙霞，刁天喜．癌症疫苗产业市场的现状与发展趋势[J]．中国新药杂志．2008，17(19)：1641—1643．
[3]McArdle SE，Rees RE．Tumor immunology：new perspectives[J]l Cancer Res，2009，69f18)：7157—7159．
[4]Kenneth Lundstrom．Alphaviruses in Gene Therapy[J]lViruses，2009，1《1)，13—25．
[5]Schlesinger S．A~havirus vectors：development mad potential therapeuticapplications．Expert Opin Biol Ther，2001，1(2)：177—191．
[6]Jonathan O R，Sergey AD，Kurt IK．A~havirus vectors and vaccination[J]l RevMed Virol,2002，12：279—296．
[7]余云舟，孙志伟，刘志刚等．基于DNA的SemlNi森林病毒复制子载体体内外高水平表达外源基因[J]l生物化学与生物物理进展．Progress in Biochemistryand Biophysics，2006，33(1)：87—94．
[8]Sun Y，D Liu，et aL A prime_boost vaccination~rNegy using a Seml~i Forestvirus replicon ve~ored DNA vaccine followed by a recombinant adenovirus protectspigs from classical swine fever[J]l Sheng Wu Gong Cheng Xue Bao．2009，25《5)：679—685．
[9]Quintaxm—Vazquez D，Vazquez—Blomquist DM，Galban Rodriguez E，et aL Avaccination strategy consisting of Semliki—Forest—virus fSFVl DNA prime andfoWlDOX—virus boost significantl,／DrotectS mice from a recombinant rHIV一1)-vaccinia-virus infection[J]l Biotechnol Appl Biochem，2005，41《1)：59-66．
[10]Durso R．J，Andjelic S，Gardner J．E et a1．Novel alphavirus vaccine encodingprostate-specific membrane antigen elicits potent cellular and humoral immuneresponses．Clin．Cancer Res．2007，13，3999-4008．[1 1]Gupta P K，SS Dahiya,et al Sindbis virus replicon-based DNA vaccine encodingRabies virus glycoprotein elicits specific humoral and cellular immune response indogs[J]l Acta Vir01．2009，53(2)：83-88．
[12]Riezebos-Brilman A_Regts J，Chen M，et al Augmentaion ofalphavirusvector--induced human papilloma virus--specific immune and anti--tumour responses bycoexpression ofinterleukin-12．Vaccine 2009，27，701-707．
[13]Gabler M，Scheiblhofer S，Kern K，et a1．Immunization with a low-dose repliconDNA vaccine encoding Phl P 5 effectively prevents allergic sensitization[J]．JAllergy Clin Immunol,2006，1 18(3)：734-41．
[14]David I．Bernsteina，Elizabeth A．Reapb，Kevin Katenb,et a1．Randomized,double-blind，Phase 1 trial ofan alphavirus replicon vaccine for cytomegalovirus inCMV seronegative adult volunteers[J]l Vaccine，20 10，28：484-493．
[15]罗颖．Survivin多肽疫苗治疗血液系统恶性肿瘤的研究进展[J]．重庆医科大学学报，2007，32(10)：1118-1120．
[16]杜冠辉，谭金风，吴淑金，李强Survivin与肿瘤的关系[J]l医学综述，2008，14 f51：658-660．
[17]Marioni G，ED’Alessandro，et aL Survivin multifaceted activity in head and neckcarcinoma：current evidence and future therapeutic challenges[J]．Acta Otolaxyng01．2010，130(1)：4-9．
[18]Tetsuhiro T，Yuji I，Tosei，et aL Clinical and immunological evaluation ofanti-apoptosis protein,survivin-derived peptide vaccine in phase I clinical study forpatients with advanced or recurrent breast cancer[J]l J Transl Med．2008，6(24)：1-1 1．
[19]Miyazaki A_J Kobayashi，et aL Phase I clinical trial of survivin-derived peptidevaccine therapy for patients with advanced or recurrent oral cancer[J]l Cancer Sci201 1，102(2)：324-329．
[20]Cole LA．Biological functions of hCG and hCG-related molecules[J]．ReprodBiol Endocrino．2010．1 8：102．
[21]李明清，唐传玲．抗hCG疫苗在肿瘤免疫治疗中的价值[J]．中国免疫学杂志，2008，24(12)：1149-1151．
[22]Qing Dong Guan,Ying Wang，Yi Wei Chu，chu．The distinct effects of threetandem repeats of C3d in the immune responses against tumor-associated antigenhCG by DNA immunization[J]l Cancer Immunol Immunother．2007，56：875-884．
[23]Xiangbing，H．，Z．Yankai，el a1．The fusion protein of HSP65 with tandemrepeats ofbeta-hCG acting as a potent tumor vaccine in suppressing hepatocarcinoma[J]l Int Immunopharmac01．2010，10(2)：230-238．
[24]Gupta R，Emens LA．GM-CSF-secreting vaccines for solid tumors：movingforward[J]．Discov Med．20 10．10(50)：52-60．
[25]Burke JM．GM-CSF-alTned replication-competent viruses for cancer[J]lCytokine Growth Factor Rev．2010．21(2-3、：149-151
[26]Clive KS，Tyler JA，Clifton GL el al Use of GM-CSF as an adjuvant withcancer vaccines：beneficial or detrimental[J]?Expert Rev Vaccines．20 10，9(5)：5 19-525．
[27]Arnett HA，JL Viney．Recent developments in IBD and the B7 family ofcostimulatory molecules[J]l Curr Opin Investig Drugs．2009，10(5)：443-45 1．
[28]Petroff MG A Perchellet．B7 fanljly molecules as regulators of the maternalimmune system in pregnancy[J]l Am J Reprod Immun01．2010，63(6)：506-519．
[29]Loos M，DM Hedderich，el a1．B7-h3 and its role in antitumor immunity[J]l ClinDev Immun01．2010，683875．
[30]Webster ML，G Zhu，el a1．Fc-independent phagocytosis；implications forintravenous IgG therapy in immune thrombocytopenia[J]．Cardiovasc HematolDisord Drug Targets．2008，8(4)：278-282．[3 1]Anthony RM，JV Ravetch．A novel role for the IgG Fc glTy’can；theanti-inflammatory activity of sialylated IgG Fcs[J]l J Clin Immun01．2010，30 Suppl 1：S9-14．
[32]Lejeune J，G Thibault，el a1．Implications of receptors for the Fc portion of IgG《FcgammaRs)in mechanism of action oftherapeutic antibodies[J]l Bull Cancer．20 10，97f51：511-522．
[33]Sesarman A，G Vidarsson,el a1)．The neonatal Fc receptor as therapeutic targetin IgG-mediated autoimmune diseases[J]l Cell Mol Life Sci．2010，67(15)：2533-2550．
[34]Ferrone C，Perales M，Goldberg S，el aL Adjuvanticity ofplasmid DNA encodingcytokines fused to immunoglobulin Fc domains[J]．Clin Cancer Res，2006，12(18)：5511-5519．
[35]Sh oji Hosaka E，Kobayashi Y，Wakitani M，el a1．Enhanced Fc dependent cellularcytotoxicity of Fc fusion proteins derived from TNF receptor II and LFA-3 by fucoseremoval fromAsh linked oligosaccharides[J]．J Biochem,2006，140《6)：777-783．
[36]FujjIa M，Y Jigami．Lipid remodeling of GPI-anchored proteins and its function[J]．Biochim Biophys Acta．2008，1780(3)：410-420．
[37]Fujita M，T Kinoshita．Structural remodeling ofGPI anchors during biosynthesisand after attachment to proteins[J]l FEBS Lett．2010，584(9)：1670-1677．
[38]Heller LC，Ugen K，Heller R．Electroporation for targeted gene transfer[J]l Exp．Opin．Drug Deliv,2005，2(2)：255-268．
[39]Luckay A_Sidhu M K，Kjeken R，et aL Effect of plasmid DNA vaccine desingand in vivo electroporation on the resulting vaccine-specific immune responses inrhesus macaques[J]l J．Virol,2007，81《10)：5257-5269．
[40]Hooper J，Golden J，FeHo A，et aL Smallr}ox DNA vaccine delivered by novelskin electroporation device protects mice against intranasal poxvirus challenge[J]．Vaccine，2007，25(10)：1814-1823．
[41]Neumann E，Schacfer-Ridder M，Wang，et al Gene transfer into mouse lyomacells by electroporation in high electric fields[J]．EMBO J，1982，《1)：841-845．
[1]Kenneth Lundstrom．Alphaviruses in Gene Therapy[J]lViruses，2009，1(1)，13—25．
[2]王红仁，李明远．基于甲病毒的RNA复制子疫苗[J]l微生物学免疫学进展，2007，35f2)：72—75．
[3]余云舟，孙志伟，刘志刚等．基于DNA的Semliki森林病毒复制子载体体内外高水平表达外源基因[J]．生物化学与生物物理学进展，2006，33(1)：87—94．
[4]王昌青，杨兵．DNA疫苗研究进展[J]l中国畜牧兽医，2009，36(12)：143—147．
[5]Ledwith BJ，Manam S，Troilo PJ，et a1．Plasmid DNA vaccines：assay forintegration into host genomic DNA[J]l Dev Biol(Basel)，2000，104：33—43．
[6]Wang Z，TroiloP J，Wang X，el a1．Detection of integration of plasmid DNA intohost genomic DNA following intramuscular injection and electroporation[J]．GeneTher,2004，11(8)：711-721．
[7]Gupta P K，SS Dahiya，el al Sindbis virus replicon-based DNA vaccine encodingRabies virus glycoprotein elicits specific humoral and cellular immune response indogs[J]l Acta Virok 2009，53(2)：83—88．
[8]Sun Y，D Liu，el a1．A prime—boost vaccination strategy using a Semliki Forestvirus replicon vectored DNA vaccine followed by a recombinant adenovirus protectspigs from classical swine fever[J]l Sheng Wu Gong Cheng Xue Bao．2009，25(5)：679—685．
[9]Riezebos—BrilmanA，RegtS J，Chen M，el a1．Augmentation ofall)havirusvector--induced human papilloma virus--specific immune and anti--tumour responses bycoexpression ofinterleukin-12．Vaccine 2009，27，701-707．
[10]M／i~itt／i A．M，MSJcinen K，Ketola A_el a1．Replication competent SemlNi Forestvirus prolongs survival in experimental lung cancer．Int．J．Cancer 2008，123，1704—171 1．
[11]Quinn,K．Effect ofintranasal administration ofSemliki Forest virus recombinantparticles expressing interferon-[3 on the progression of experimental autoimmuneencephalomyelitis．M01．Med．Rep．2008，1，335—342．
[12]Zheng M，N Jin,el a1．Immunogenicity and protective efficacy of Semliki forestvirus replicon-based DNA vaccines encoding goatpox virus structural proteins[J]lVirology．2009，391(1)：33—43．
[13]Lundstrom,K．Biology and application ofalphaviruses in gene therapy[J]l GeneTher．2005，Suppl．1，$92—97．
[14]Durso R．J，Andjelic S，Gardner J．P，el a1．Novel alphavirus vaccine encodingprostate—specific membrane antigen elicits potent cellular and humoral immuneresponses．Clin．Cancer Res．2007，13，3999—4008．
[15]Leitner w_Hwang L，Bergmann L，el a1．Apoptosis is essential for the increasedefficacy of alphaviral replicase—based DNA vaccines[J]l Vaccine，2004，22(1 1-12)：1537—1 544
[1]Ambrosini G，Adida C，Altieri D．el aL A novel an apoptosis gene，Survivin,expressed in cancer and lymphoma[J]．Nat Med，1997，3(8)：917—921．
[2]罗颖．Survivin多肽疫苗治疗血液系统恶性肿瘤的研究进展[J]l重庆医科大学学报，2007，32(10)：1118-1120．
[3]杜冠辉，谭金风，吴淑金，李强Survivin与肿瘤的关系[J]l医学综述，2008，14 (5)：658—660．
[4]Marioni G，ED’Alessandro，el aL Survivin multifaceted a~ivity in head and neckcarcinoma：current evidence and future therapeutic challenges[J]l Acta Otolaryng01．2010，130(1)：4—9．
[5]Ulf-Hakan S，Henrik A_Kristina H．Human chorionic gonadotropin in cancer[J]lClinical Biochemistry 2004，(37)：549—561．
[6]Cole LA．Biological fi．mctions of hCG and hCG—related molecules[J]l ReprodBiol Endocrino．2010．1 8：102．
[7]李明清，唐传玲．抗hCG疫苗在肿瘤免疫治疗中的价值[J]．中国免疫学杂志，2008，24(12)：1149—1151．
[8]王永芬，马辉，吴学军．抗人绒毛膜促性腺激素单克隆抗体的制备与鉴定[J]l细胞与分子免疫学杂志2008，24(1 1)：1 102—1 103．
[9]Tetsuhiro T，Yuji I，Tosei．el al Clinical and immunological evaluation ofanti-apoptosis protein,survivin—derived peptide vaccine in phase I clinical study forpatients with advanced or recurrent breast cancer[J]l J Tramsl Med．2008，6(24)：1-1 1．
[10]Yamg Z，L Wang，el aL A novel mimovirus vaccine containing survivin epitopewith adjuvant IL一15 induces long—lasting cellular immunity and high antitumorefficiency[J]．Mol Immun01．2008，45(6)：1674—1681．
[11]Li H，MH Li，el aL Human survivin modified DCs vaccine inhibits laryngealcancer in vivo and in vitro[J]．Zhonghua Er Bi Yah Hou Tou Jing Wai Ke Za Zhi2009，44f9)：767—771．
[12]Miyazaki A_J Kobayashi，et aL Phase I clinical trial of survivin-derived peptidevaccine therapy for patients with advanced or recurrent oral cancer[J]．Cancer Sci2011，102(2)：324—329．
[13]Xiangbing H，Z Yankai，et aL The fusion protein ofHSP65 with tandem repeatsof beta—hCG acting as a potent tumor vaccine in suppressing hepatocarcinoma[J]l IntImmunopharmacol 2010，10(2)：230—238．
[14]P．J．Delves，R．K．Iles，I．M．Roitt，et a1．Designing a new generation ofanti—hCGvaccines for cancer therapy[J]l Molecular and Ce~ular Endocrinology 2007(260-262)：276-281．
[15]He Yi，Yaxl Rong，Zhang Yankai，et aL Improved efficacy of DNA vaccinationagainst breast cancer by boosting with the repeat beta-hCG C—terminal peptide carriedby mycobacte heat—shock protein HSP65[J]．Vaccine 2006，24(14)：2575—2584．
[16]Rong X，Noriko M，Yunping L．et aL DNA Vaccine Targeting SurvivinCombines Apoptosis with Suppression of Angiogenesis in Lung Tumor Eradication[J]l Cancer Res 2005，65(2)：553—561．
[17]Guan QD，Wang Y，Chu Yw，eL The distinct evects ofthree tandem repeatsof C3d in the immune responses against tumor—associated antigen hCGB by DNAimmunization[J]．Cancer Immunol Immun 2007，56(6)：875—884．
[18]Kitamura H，Torigoe T，Honma I．et aL expression and antigenicity of survivin,an inhibitor of apoptosis family member，in bladder cancer：implications for specificimmunotherapy[J]．Urology，2006，67(5)：955—959．
[19]Abad JD，Wrzensinski C，Overwijk W，et aL T—cell receptor gene therapy ofestablishedtumorsin amurinemelanomamodel[J]l JImmunother，2008，31(1)：1-6．
[20]Guimaraes Ferreira CA，Rodrigues EG，Mortara RA，et aL Antitumor effects invitro and in vivo and mechanisms ofprotection against melanoma B16F10一Nexcellsby fastuosain，a cysteine proteinase fcom Bromelia fastuosa[J]l Neoplasia,2007，9(9)：723．733
[1]Koyama S，T Kawagoe．et aL Immunological mechanism ofDNA vaccine efficacy[J]l Tanpakushitsu Kakusan Koso 2009，54(8 Suppl)：1096—1100．
[2]Jana B，Milada H，Michal K．et aL DNA vaccines：are they still just a powerfultool for the future[J]?Arch．Immun01．Ther．Exp．2007，55，387—398
[3]lJu S，Wang S，Jill M，et aL Current progress of DNA vaccine studies in humans[J]l Expert Rev．Vaccine，2008，7(2)：175—191．
[4]Wu TC．Therapeutic human papillomavirus DNA vaccination strategies to controlcervical cancer[J]．Eur．J．Immun．2007，37(2)：310—314．
[5]Bedognetti D，E．Wang，et aL Gene—expression profiling in vaccine therapy andimmunotherapy for cancer[J]l Expert Rev Vaccines．2010，9(6)：555—565．
[6]Kieback，E，W．Uckert．Enhanced T cell receptor gene therapy for cancer[J]．Expert Opin Biol Ther．2010，10(5)：749—762．
[7]Samaranayake，H，A．M．Maatta，et aL Future prospects and challenges ofantiangiogenic cancer gene therapy[J]l Hum Gene Ther．2010，21(4)：381—396．
[8]Marhsall E．Gene therapy death prompts review of adenovirus vector[J]l Science，2000，286：2244—2245．
[9]Mammohan S，Aravind C，Derek OH，et aL Nanopaxticles and micropazticles asvaccine delivery systems[J]l Expert Rev．Vaccines，2007，6(5)：797—808．
[10]何保凌，孙恩杰，黄文．基因枪技术及其在基因治疗中的应用进展[J]l生物学杂志2007，24(2)：47—50．
[10]Fuller D，Loudon P，SchmaUohn C．Preclinical and clinical progress ofparticle—medi~ed DNA vaccines for infectious diseases[J]l Methods，2006，40(1)：86—97．
[11]Sharpe M，Lynch D，Topham S，et aL Protection of mice from H5N1 influenzachal~nge by prophylactic DNA vaccination using particle mediated epidermaldelivery[J]l Vaccine，2007，25(34)：6392—6398．
[12]Neumann E，Schacfer-Ridder M，Wang，et al Gene transfer into mouse lyomacells by electroporation in high electric fields[J]l EMBO J，1982，(1)：841—845．
[13]Ionescu—zanetti C，Blatz A_Khine M，et aL Electrophoresis—assisted single—cellelectroporation for efficient intracellulax delivery[J]．Biomed Micro devices，2008，10(1)：113—116．
[14]Luckay A_Sidhu M K，Kjeken R，et aL Effect of plasmid DNA vaccine desingand in vivo electroporation on the resuking vaccine—specific immune responses inrhesus macaques[J]l J．Virol,2007，81(10)：5257—5269．
[15]Hooper J，Golden J，FeHo A，et aL Smallr}OX DNA vaccine delivered by novelskin electroporation device protects mice against intranasal poxvkus chal~nge[J]lVaccine，2007，25(10)：1814—1823．
[16]Valero A_Post JN，Van Nieuwkasteele Jw et aL Gene transfer and proteindynamics in stem cells using single cell electroporation in amicrofluidic device[J]lLab Chip，2008，8(1)：62—67．
[1]DouganM，G Dranofl：Immunetherapy for cancer[J]l Annu RevImmun01．2009，27：83—117．
[2]Pellegrini，M，Tw Mak．Tumor immune therapy：lessons from infection andimplications for cancer—can IL-7 help overcome immune inhibitory networks[J]?Eur J Immun01．2010，40(7)：1852—1861．
[3]Ikeda H，N Imai，el al Cancer treatment with gene—modified T cells or DNAvaccine[J]l Nippon Rinsho 2010，68(Suppl 8)：634—640．
[4]Becker J．L BM Olson,el al DNA vaccine encoding prostatic acid phosphatase(PAP)elicits long—term T-cell responses in patients with recurrent prostate cancer[J]lJ Immuno ther．2010，33(6)：639—647．
[5]Sedgwick JD．ELISPOT assay：a personal retrospective．Methods Mol Biol,2005，302(1)：3—14．
[6]Wolff JA，Malone RW,Williams P，el al Direct gene transfer into mouse musclein vivo[J]l Science，1990，247(49)：1465—1468．
[7]Koyama S，T Kawagoe，el al Immunological mechanism of DNA vaccineefficacy[J]l Tanpakushitsu Kakusan Koso 2009，54(8 Supp~：1096—1100．
[8]Sham L，Shixiaw，JillM，el a1．Charrent progressofDNA vaccine studiesin humans[J]l Expert Rev．Vaccines,2008，7(2)：175—191．
[9]Jaxm B，Milada H，Michal K，el a1．DNA vaccines：are they stilljust a powerful toolforthefuture[J]?Arch．Immunol,Ther．Exp．2007，55：387-398．
[10]Xuezw，XLZhmt Research progress onDNAtulTlor vaccine[J]．Zhongguo YiMlao He Mlan Y1．2U10，16(2)：183—186．
[11]WuWC，DYjin,etal．Inductionofaxltigen-specificCTL and antibody responsesinmice by a novel recombinant tandem repeat DNA vaccine targeting at mucin 1 ofpancreatic cancer[J]．J Cancer Res．Clin Oncok 2010，136(12)：1861—1868．
[12]Chain RC，B．Gutierrez，el aL Enhancement ofDNA cancer vaccine efficacy bycombination with anti-angiogenesis in regression of estab~shed subcutaneous B16melanoma[J]．Oncol Rep 2009，22(5)：1 197—1203．
[13]Lai MD，MC Yen,el aL The effects of DNA formulation and administration routeon cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung canceranimal model[J]．Genet Vaccines Ther．2009，7：2．
[14]Pakravam N_L Lamgroudi，el aL Co—adminiaration of GP96 and Her2／neu DNAvaccine in a Her2 breast cancer model[J]．Cell Stress Chaperones．2010，15(6)：977—984．
[15]McNeel DG，EJ Dunphy，el aL Safety and immunological efficacy of a DNAvaccine encoding prostatic acid phosphatase in patients with stage DO prostate cancer[J]．J C1in Oncok 2009，27(25)：4047—4054．
[16]Becker JT，BM．Olsork el aL DNA vaccine encoding prostatic acid phosphatase(PAP)elicits long-term T—cell responses in patients with recurrent prostate cancer[J]．JImmunother-2010，33(6)：639—647．
[1]Kenneth Lundstrom．Alphaviruses in Gene Therapy[J]lViruses，2009，1(1)，13—25．
[2]Schlesinger S．A~havkus vectors：development and potential therapeuticapplications．Expeg Opin Biol Ther，2001，1(2)：177—191．
[3]Jonathan O R，Sergey AD，Kurt IK．A~havirus vectors and vaccination[J]l RevMed Virol,2002，12：279—296．
[4]David I．Bernsteina,Elizabeth A．Reapb，Kevin Katenb,el aL Randomized，double—blind，Phase 1 trial ofan alphavirus replicon vaccine for cytomegalovirus inCMV seronegative adult volunteers[J]l Vaccine，2010，28：484—493．
[5]Lundstrom,K．Biology and application of alphaviruses in gene therapy[J]l GeneTher．2005，Suppl．1，$92—97．
[6]Durso R．J，Andjelic S，Gardner J．P et a1．Novel alphavirus vaccine encodingprostate—specific membrane antigen elicits potent cellular and humoral immuneresponses．Clin．Cancer Res．2007，13，3999—4008．
[7]Gupta P K，SS Dahiya，et al Sindbis virus replicon-based DNA vaccine encodingRabies virus glycoprotein elicits specific humoral and cellular immune response indogs[J]．Acta Virol 2009，53(2)：83—88．
[8]Saxena S，SS Dabiya，el aL A sindbis virus replicon-based DNA vaccine encodingthe rabies virus glycoprotein elicits immune responses and complete protection inmice from~thal chal~nge[J]．Vaccine 2008，26(51)：6592—6601．
[9]Zheng M，N Jin,et aL Immunogenicity and protective efficacy of Semliki forestvirus replicon-based DNA vaccines encoding go．pox virus structural proteins[J]lVirology．2009，391(1)：33—43．
[10] Li N，YZ Yu，et aL Enhancement of the immunogenicity of DNA repliconvaccine of Clostridium botulinum neurotoxin serotype A by GM—CSF gene adjuvant[J]l Immunopharmacol Immunotoxic01．201 1，33(1)：21 1-219．
[11]Yu Y J Yu, eL Individual and bivalent vaccines against botulinum neurotoxinserotypes A and B using DNA-based Semliki Forest virus vectors[J]l Vaccine．2009，27(44)：6148—6153．
[12]Sun Y N Li，et aL Enhanced immunity against classical swine fever in pigsinduced by prime—boost immunization using an alphavirus replicon-vectored DNAvaccine and a recombinant adenovirus[J]l Vet Immunol Immunopath01．2010，137(1—21：20—27．
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |