基于日本脑炎病毒复制子载体系统的疫苗研究
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摘要
乙型脑炎病毒(Japanese encephalitis virus,JEV)属于黄病毒科黄病毒属,主要在亚洲流行,可引起严重的中枢神经系统传染病,感染后致死率高达30%左右,并有50%的存活者会留下永久性的神经系统后遗症。目前国际通用的灭活疫苗存在着副作用较大且费用较高的问题,而我国应用的减毒活疫苗未通过国际认证且存在着一定的回复风险,因此对于乙型脑炎疫苗的研究工作依然十分重要。
复制子表达系统是在RNA病毒感染性克隆的基础上删除病毒的结构基因,亚克隆其它外源基因,利用(+)RNA病毒的自主复制特性,转染细胞后表达自身非结构蛋白以及外源蛋白。如果辅以表达病毒结构蛋白的辅助载体共转染细胞,可以重新包装成新的假病毒颗粒。理论上讲,复制子RNA在细胞胞浆内大量扩增,使得外源基因也随之高水平表达,是表达异源基因的良好载体。假病毒颗粒只具有“一次感染能力”,因为其包装的RNA是去除了结构基因的复制子,感染细胞后由于缺乏病毒的结构蛋白,不能够重新包装成为病毒颗粒,不会感染周围细胞,作为疫苗是安全的。近年来,KUNV、TBEV和YFV复制子载体系统已经被报道可以作为发展新型疫苗的潜在的有用的工具。我们希望构建JEV复制子载体系统,并将其开发成为疫苗研究的新平台。
我们在JEV疫苗株SA14-14-2基础上,经过优化构建了一系列复制子载体。分别将绿色荧光蛋白(EGFP)和LacZ报告基因插入复制子载体pCMW-2M、pCMW-G2R中。pCMW-2MEG转染BHK-21细胞,24小时后可以观察到EGFP的表达,在转染72小时后更为明显,在转染7天后也能观察到。流式细胞术定量检测EGFP复制子转染BHK-21细胞后有10%左右的阳性率。同样,含Lac Z的复制子转染细胞后也可获得报告基因的表达。进一步为检测复制子的体内功能活性,我们将含Lac Z报告基因的复制子载体直接进行乳鼠脑内注射,结果显示在小鼠脑组织广泛表达Lac Z,阳性信号强,覆盖面积广。这些结果表明了JEV复制子pCMW-2M能够有效地复制并高效表达外源蛋白,为建立JEV复制子载体系统打下良好基础。
建立稳定表达结构蛋白的细胞系是复制子系统用于获得假病毒颗粒的重要环节,细胞系的成功建立能够使收获假病毒颗粒的实验步骤简化,并且获得的病毒量较多,进一步放大培养也成为现实。我们通过实验克服了E蛋白对宿主细胞的毒性,建立了稳定表达JEV结构蛋白的细胞系,可包装复制子产生假病毒颗粒,这是国内外首次关于JEV包装细胞系的报道。
在先期实验中,我们尝试构建表达prM-E蛋白的细胞系,但是由于E蛋白对宿主细胞的毒性大,挑选不到有效表达prM-E蛋白的细胞克隆。为解决这一问题,我们先后进行了真核表达载体的改造、更换表达效率低的真核表达载体、构建在表达基因前添加信号肽序列的载体、构建了改变prM-E基因FURIN切割位点的真核表达载体等工作,但是都没有取得满意的效果。由于整体结构蛋白的完整性对于结构蛋白的翻译后加工有促进作用,添加JEV C基因可能对结构蛋白的表达起稳定作用,并且能进一步促进病毒的包装。因此我们构建了含有完整JEV结构基因的质粒pcJECME,转染BHK-21细胞后挑取细胞克隆,经过ELISA、Westren Blot、IFA的鉴定,最终获得了稳定表达JEV结构蛋白的细胞系,经过100代次以上的培养,仍有50%以上的细胞表达目的蛋白。用复制子载体pCMW-2MLac转染该细胞克隆,Lac Z的表达能力有了明显的提高。
利用复制子系统获得假病毒颗粒的方式有两种,一种是复制子载体和表达结构蛋白的真核表达载体共转染敏感细胞;另一种是复制子载体转染稳定表达结构蛋白的细胞系。由于前者操作较为复杂且获得的假病毒滴度较低,我们利用已经构建好的JEV结构蛋白细胞系,包装JEV复制子载体以获得只具有一次感染能力的JEV假病毒颗粒。通过电镜及RT-PCR的方法鉴定了复制子转染包装细胞系后可产生假病毒颗粒,滴度可达到1.6×105 U/ml。将其免疫小鼠后,血清特异性抗E蛋白抗体滴度可达到1:2560,中和抗体滴度可达1:128。可对73%的小鼠保护免受病毒攻击。以上实验结果均表明JEV假病毒颗粒具有良好的免疫原性。
由于假病毒颗粒疫苗具有很好的应用前景,我们首次将JEV复制子载体系统用于炭疽疫苗的研究。炭疽杆菌(Bacillus anthracis)是一种能形成芽胞的革兰氏阳性菌,由它感染引起的炭疽病是严重危害人类的一种致死性的重大传染病,同时它又是一种潜在的生物战剂,炭疽疫苗的研究对于生物反恐具有重要的意义。PA是炭疽毒素的三大组分之一,是引起机体免疫应答的最有效的免疫原,单独应用没有毒性,当前应用的疫苗主要成分是PA。将构建好的含炭疽PA4基因的JEV复制子转染包装细胞系,经过制备和纯化可得到病毒滴度为5×105U/ml的假病毒颗粒,免疫小鼠后能产生特异性的抗体,在免疫二次后的抗体滴度为1:1280,进一步验证了JEV复制子载体系统开发研究新型疫苗是可行的。
由于JEV E蛋白可诱导产生保护性中和抗体并对病毒毒力起重要作用,是重要的抗原成份。我们还表达纯化了JEV E蛋白结构域Ⅲ,研究其作为亚单位疫苗预防JEV感染的可能。高效可溶性表达了融合蛋白Trx-JEDⅢ,表达量约占菌体蛋白的75%。利用纯化的JEDⅢ蛋白免疫新西兰兔,获得了高达1:4×105滴度的抗JEV抗体;免疫BALB/C鼠,获得了1:8×104滴度的抗JEV抗体和1:2×103滴度的抗YFV抗体。免疫小鼠血清的中和抗体可达1:256。实验表明JE DIII蛋白的抗原性及免疫原性较好,有开发研制成亚单位疫苗的潜能。
综上所述,本研究建立了稳定的JEV复制子载体系统,可在体内外高效表达EGFP、LacZ报告基因。构建了高效稳定表达结构蛋白的细胞系,包装复制子后可产生1.6×105 U/ml的假病毒颗粒,具有良好的免疫原性,并获得了PA-JEV假病毒颗粒,免疫小鼠后可获得特异性抗体。总的来说,我们首次建立了JEV复制子载体系统的技术平台,为今后开发研究多种新型疫苗奠定了坚实的基础。
Japanese encephalitis virus (JEV) is an emerging threat throughout Southern and Southeast Asia and Australia. Although most JEV infections are asymptomatic, the estimated 0.3% that lead to disease result in over 35,000 cases including 10,000 deaths annually worldwide, and many of the remaining cases produce permanent sequelae. Japanese encephalitis virus is one of globally important human pathogens. Despite an emergence and resurgence of JEV-mediated disease,specific therapies are not yet available.
Subgenomic replicons of positive-stranded RNA viruses , which contain all the non-structural proteins required for amplifying themselves but lack some or all of the structural proteins,have been shown to be useful tools to study viral replication in the absence of virion assembly and maturation。Several of the flaviviruses, including JEV, display a number of characteristics that make them useful for the development of delivery vectors to express foreign genes。In the past few years, heterologous genes have been expressed in the context of (i) an infectious virus genome, in the case of WNV and TBEV; or (ii) a replicon lacking viral structural proteins (C, prM, and E), in the case of several flaviviruses, including DENV,YFV, WNV,KUNV and TBEV. JEV has not been used for this purpose to date. Here we report three strategies for utilizing JEV replicon vectors to express foreign genes in a variety of cell types, taking advantage of a full-length infectious JEV cDNA that we have previously constructed. These JEV replicon expression systems can potentially serve as powerful tools in both in vitro and in vivo applications. The large packaging capacity of JEV offers a distinct advantage with regard to expressing larger genes and the treatment of associated diseases.
We report the construction of subgenomic replicons derived from a full-length cDNA clone of the SA14-14-2 strain of JEV. Using multiple strategies, various reporter genes including green fluorescence protein (GFP) and Lac Z were expressed from these replicons in a DNA replication-dependent manner. That expressed the JEV structural proteins were also constructed and could be used to package JEV replicons into pseudo-infectious virus-like particles (PIPs). We inserted enhanced green fluorescent protein (EGFP) and Lac Z reporter gene into JEV replicon pCMW-2M, pCMW-G2R respectively,expressions of EGFP in BHK -21 cells transfected with pCMW-2MEG were determined from 24h post-transfection. EGFP was observed in cytoplasm. EGFP positive cells were seen at 72h post-transfection, whereas the expression was very few at 24h post-transfection. These results indicate that pCMW-2MEG can express foreign gene of EGFP. Just like these, Expression of the Lac Z gene could be detected at 4 days after transfection.
The 53-kDa envelope (E) glycoprotein of JEV has an important role in virus adhesion and entry into target cells through receptor binding and, therefore, in inducing neutralizing antibodies that protect hosts against JEV infection. The higher expression of the E-VLP antigen should be more toxic to the expressing cells, resulting in cell fusion, apoptosis, and death.The presence of the capsid protein was found to be absolutely required for packaging. Moreover, the ability to efficiently package JEV replicons indicates that any potential RNA sequences within JEV genome, which might function as packaging signals, are not contained within the JEV structural protein coding sequence.
Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vectors. The recombinant plasmid pCJEME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJECME, the cell lines continuously expressing structural proteins can be selected with G418. And the expression products of pCJECME vector could be detected by ELISA,Western Blot and IFA assay. It shows that the JEV capsid protein can enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.
To facilitate our future investigations of virus assembly, expressing the JEV structural proteins were constructed and used to package JEV replicons into PIPs. With the application of recombinant DNA technology and reverse genetics, PIPs of several flaviviruses thus established have been shown to be valuable for studying viral replication and inhibitors and promising for developing vectors of gene therapy and vaccines。In this study, we have established a cell line stably producing structural proteins of JEV, which can successfully incorporate JEV replicon and produce single-round infectious PIPs. A closer examination of the expression of replicon and the infectivity of PIPs containing replicon revealed that the infectivity of PIPs correlated with the expression of the replicon, The highest titer of JE PIPs in this study, was 1.6×105 U/ml.After immune with JE PIPs induced specific antibody after immunization of mice.The antibody titers reached to 1:2560 after the twice immunization,Further improvement in the efficiency of RNA transfection is needed to increase the titers of our PIPs.
The fetal disease anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis, and Bacillus anthracis has been postulated to be a potential agent of biowarfare and bioterrorism. PA is the main composition of current vaccine and the most effective immunogen to induce immune response. The fourth domain of PA gene was cloned into JEV replicon DNA vector, then transfect packaging cell lines with and obtain the PIPs with high titers of 5×105U/ml. The PIPs can infect cell in vitro and induced 1:1280 titer antibody after immunization of mice.The PA4 PIPs based on JEV replicon are prominsing candiant vaccines for human use and establish the foundation for deep reseach of multi-antigen vaccine of anthrax.
Here we report the development of a JEV-based vector system for foreign gene expression that utilizes a full-length infectious JEV cDNA. involved a variety of replication-competent, propagation-deficient viral replicon vector DNAs showing a spectrum of DNA replication efficiencies. The other procedure made use of stable packaging cell lines for the production of single-round infectious, propagation-deficient JEV PIPs. These data strongly suggest that our JEV replicon vector systems represent attractive potential candidates for foreign gene expression and antiviral compound screening in a wide variety of cells in vitro,and possibly in vivo for immunization applications.
The E protein of Japanese encephalitis virus (JEV) is the major antigen used to elicit neutralizing antibody response and protective immunity in hosts. In this study, the domain III protein of the attenuated strain SA14-14-2 was cloned to the pET32a expression vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The recombinant protein was unique in forming a large fraction of the soluble recombinant protein in E. coli. The antigenicity and immunogenicity of the purified DⅢprotein was tested by Western Blot analysis .The purified domain III protein was emulsified in Freund’s adjuvant (FA) for immunization in mice and rabbits. After 3 times immunized,Rabbits’serum generated 1:4×105 anti-JEV antibody titers .Mice’serum generated 1:7×104 anti-JEV antibody titers and1:2×103 anti-YFV antibody titers .Then resulted in eliciting neutralizing antibodies and protective immunity in ICR mice. These studies can provide useful information for further developing the domain III recombinant protein vaccine against JEV.
复制子表达系统是在RNA病毒感染性克隆的基础上删除病毒的结构基因,亚克隆其它外源基因,利用(+)RNA病毒的自主复制特性,转染细胞后表达自身非结构蛋白以及外源蛋白。如果辅以表达病毒结构蛋白的辅助载体共转染细胞,可以重新包装成新的假病毒颗粒。理论上讲,复制子RNA在细胞胞浆内大量扩增,使得外源基因也随之高水平表达,是表达异源基因的良好载体。假病毒颗粒只具有“一次感染能力”,因为其包装的RNA是去除了结构基因的复制子,感染细胞后由于缺乏病毒的结构蛋白,不能够重新包装成为病毒颗粒,不会感染周围细胞,作为疫苗是安全的。近年来,KUNV、TBEV和YFV复制子载体系统已经被报道可以作为发展新型疫苗的潜在的有用的工具。我们希望构建JEV复制子载体系统,并将其开发成为疫苗研究的新平台。
我们在JEV疫苗株SA14-14-2基础上,经过优化构建了一系列复制子载体。分别将绿色荧光蛋白(EGFP)和LacZ报告基因插入复制子载体pCMW-2M、pCMW-G2R中。pCMW-2MEG转染BHK-21细胞,24小时后可以观察到EGFP的表达,在转染72小时后更为明显,在转染7天后也能观察到。流式细胞术定量检测EGFP复制子转染BHK-21细胞后有10%左右的阳性率。同样,含Lac Z的复制子转染细胞后也可获得报告基因的表达。进一步为检测复制子的体内功能活性,我们将含Lac Z报告基因的复制子载体直接进行乳鼠脑内注射,结果显示在小鼠脑组织广泛表达Lac Z,阳性信号强,覆盖面积广。这些结果表明了JEV复制子pCMW-2M能够有效地复制并高效表达外源蛋白,为建立JEV复制子载体系统打下良好基础。
建立稳定表达结构蛋白的细胞系是复制子系统用于获得假病毒颗粒的重要环节,细胞系的成功建立能够使收获假病毒颗粒的实验步骤简化,并且获得的病毒量较多,进一步放大培养也成为现实。我们通过实验克服了E蛋白对宿主细胞的毒性,建立了稳定表达JEV结构蛋白的细胞系,可包装复制子产生假病毒颗粒,这是国内外首次关于JEV包装细胞系的报道。
在先期实验中,我们尝试构建表达prM-E蛋白的细胞系,但是由于E蛋白对宿主细胞的毒性大,挑选不到有效表达prM-E蛋白的细胞克隆。为解决这一问题,我们先后进行了真核表达载体的改造、更换表达效率低的真核表达载体、构建在表达基因前添加信号肽序列的载体、构建了改变prM-E基因FURIN切割位点的真核表达载体等工作,但是都没有取得满意的效果。由于整体结构蛋白的完整性对于结构蛋白的翻译后加工有促进作用,添加JEV C基因可能对结构蛋白的表达起稳定作用,并且能进一步促进病毒的包装。因此我们构建了含有完整JEV结构基因的质粒pcJECME,转染BHK-21细胞后挑取细胞克隆,经过ELISA、Westren Blot、IFA的鉴定,最终获得了稳定表达JEV结构蛋白的细胞系,经过100代次以上的培养,仍有50%以上的细胞表达目的蛋白。用复制子载体pCMW-2MLac转染该细胞克隆,Lac Z的表达能力有了明显的提高。
利用复制子系统获得假病毒颗粒的方式有两种,一种是复制子载体和表达结构蛋白的真核表达载体共转染敏感细胞;另一种是复制子载体转染稳定表达结构蛋白的细胞系。由于前者操作较为复杂且获得的假病毒滴度较低,我们利用已经构建好的JEV结构蛋白细胞系,包装JEV复制子载体以获得只具有一次感染能力的JEV假病毒颗粒。通过电镜及RT-PCR的方法鉴定了复制子转染包装细胞系后可产生假病毒颗粒,滴度可达到1.6×105 U/ml。将其免疫小鼠后,血清特异性抗E蛋白抗体滴度可达到1:2560,中和抗体滴度可达1:128。可对73%的小鼠保护免受病毒攻击。以上实验结果均表明JEV假病毒颗粒具有良好的免疫原性。
由于假病毒颗粒疫苗具有很好的应用前景,我们首次将JEV复制子载体系统用于炭疽疫苗的研究。炭疽杆菌(Bacillus anthracis)是一种能形成芽胞的革兰氏阳性菌,由它感染引起的炭疽病是严重危害人类的一种致死性的重大传染病,同时它又是一种潜在的生物战剂,炭疽疫苗的研究对于生物反恐具有重要的意义。PA是炭疽毒素的三大组分之一,是引起机体免疫应答的最有效的免疫原,单独应用没有毒性,当前应用的疫苗主要成分是PA。将构建好的含炭疽PA4基因的JEV复制子转染包装细胞系,经过制备和纯化可得到病毒滴度为5×105U/ml的假病毒颗粒,免疫小鼠后能产生特异性的抗体,在免疫二次后的抗体滴度为1:1280,进一步验证了JEV复制子载体系统开发研究新型疫苗是可行的。
由于JEV E蛋白可诱导产生保护性中和抗体并对病毒毒力起重要作用,是重要的抗原成份。我们还表达纯化了JEV E蛋白结构域Ⅲ,研究其作为亚单位疫苗预防JEV感染的可能。高效可溶性表达了融合蛋白Trx-JEDⅢ,表达量约占菌体蛋白的75%。利用纯化的JEDⅢ蛋白免疫新西兰兔,获得了高达1:4×105滴度的抗JEV抗体;免疫BALB/C鼠,获得了1:8×104滴度的抗JEV抗体和1:2×103滴度的抗YFV抗体。免疫小鼠血清的中和抗体可达1:256。实验表明JE DIII蛋白的抗原性及免疫原性较好,有开发研制成亚单位疫苗的潜能。
综上所述,本研究建立了稳定的JEV复制子载体系统,可在体内外高效表达EGFP、LacZ报告基因。构建了高效稳定表达结构蛋白的细胞系,包装复制子后可产生1.6×105 U/ml的假病毒颗粒,具有良好的免疫原性,并获得了PA-JEV假病毒颗粒,免疫小鼠后可获得特异性抗体。总的来说,我们首次建立了JEV复制子载体系统的技术平台,为今后开发研究多种新型疫苗奠定了坚实的基础。
Japanese encephalitis virus (JEV) is an emerging threat throughout Southern and Southeast Asia and Australia. Although most JEV infections are asymptomatic, the estimated 0.3% that lead to disease result in over 35,000 cases including 10,000 deaths annually worldwide, and many of the remaining cases produce permanent sequelae. Japanese encephalitis virus is one of globally important human pathogens. Despite an emergence and resurgence of JEV-mediated disease,specific therapies are not yet available.
Subgenomic replicons of positive-stranded RNA viruses , which contain all the non-structural proteins required for amplifying themselves but lack some or all of the structural proteins,have been shown to be useful tools to study viral replication in the absence of virion assembly and maturation。Several of the flaviviruses, including JEV, display a number of characteristics that make them useful for the development of delivery vectors to express foreign genes。In the past few years, heterologous genes have been expressed in the context of (i) an infectious virus genome, in the case of WNV and TBEV; or (ii) a replicon lacking viral structural proteins (C, prM, and E), in the case of several flaviviruses, including DENV,YFV, WNV,KUNV and TBEV. JEV has not been used for this purpose to date. Here we report three strategies for utilizing JEV replicon vectors to express foreign genes in a variety of cell types, taking advantage of a full-length infectious JEV cDNA that we have previously constructed. These JEV replicon expression systems can potentially serve as powerful tools in both in vitro and in vivo applications. The large packaging capacity of JEV offers a distinct advantage with regard to expressing larger genes and the treatment of associated diseases.
We report the construction of subgenomic replicons derived from a full-length cDNA clone of the SA14-14-2 strain of JEV. Using multiple strategies, various reporter genes including green fluorescence protein (GFP) and Lac Z were expressed from these replicons in a DNA replication-dependent manner. That expressed the JEV structural proteins were also constructed and could be used to package JEV replicons into pseudo-infectious virus-like particles (PIPs). We inserted enhanced green fluorescent protein (EGFP) and Lac Z reporter gene into JEV replicon pCMW-2M, pCMW-G2R respectively,expressions of EGFP in BHK -21 cells transfected with pCMW-2MEG were determined from 24h post-transfection. EGFP was observed in cytoplasm. EGFP positive cells were seen at 72h post-transfection, whereas the expression was very few at 24h post-transfection. These results indicate that pCMW-2MEG can express foreign gene of EGFP. Just like these, Expression of the Lac Z gene could be detected at 4 days after transfection.
The 53-kDa envelope (E) glycoprotein of JEV has an important role in virus adhesion and entry into target cells through receptor binding and, therefore, in inducing neutralizing antibodies that protect hosts against JEV infection. The higher expression of the E-VLP antigen should be more toxic to the expressing cells, resulting in cell fusion, apoptosis, and death.The presence of the capsid protein was found to be absolutely required for packaging. Moreover, the ability to efficiently package JEV replicons indicates that any potential RNA sequences within JEV genome, which might function as packaging signals, are not contained within the JEV structural protein coding sequence.
Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vectors. The recombinant plasmid pCJEME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJECME, the cell lines continuously expressing structural proteins can be selected with G418. And the expression products of pCJECME vector could be detected by ELISA,Western Blot and IFA assay. It shows that the JEV capsid protein can enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.
To facilitate our future investigations of virus assembly, expressing the JEV structural proteins were constructed and used to package JEV replicons into PIPs. With the application of recombinant DNA technology and reverse genetics, PIPs of several flaviviruses thus established have been shown to be valuable for studying viral replication and inhibitors and promising for developing vectors of gene therapy and vaccines。In this study, we have established a cell line stably producing structural proteins of JEV, which can successfully incorporate JEV replicon and produce single-round infectious PIPs. A closer examination of the expression of replicon and the infectivity of PIPs containing replicon revealed that the infectivity of PIPs correlated with the expression of the replicon, The highest titer of JE PIPs in this study, was 1.6×105 U/ml.After immune with JE PIPs induced specific antibody after immunization of mice.The antibody titers reached to 1:2560 after the twice immunization,Further improvement in the efficiency of RNA transfection is needed to increase the titers of our PIPs.
The fetal disease anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis, and Bacillus anthracis has been postulated to be a potential agent of biowarfare and bioterrorism. PA is the main composition of current vaccine and the most effective immunogen to induce immune response. The fourth domain of PA gene was cloned into JEV replicon DNA vector, then transfect packaging cell lines with and obtain the PIPs with high titers of 5×105U/ml. The PIPs can infect cell in vitro and induced 1:1280 titer antibody after immunization of mice.The PA4 PIPs based on JEV replicon are prominsing candiant vaccines for human use and establish the foundation for deep reseach of multi-antigen vaccine of anthrax.
Here we report the development of a JEV-based vector system for foreign gene expression that utilizes a full-length infectious JEV cDNA. involved a variety of replication-competent, propagation-deficient viral replicon vector DNAs showing a spectrum of DNA replication efficiencies. The other procedure made use of stable packaging cell lines for the production of single-round infectious, propagation-deficient JEV PIPs. These data strongly suggest that our JEV replicon vector systems represent attractive potential candidates for foreign gene expression and antiviral compound screening in a wide variety of cells in vitro,and possibly in vivo for immunization applications.
The E protein of Japanese encephalitis virus (JEV) is the major antigen used to elicit neutralizing antibody response and protective immunity in hosts. In this study, the domain III protein of the attenuated strain SA14-14-2 was cloned to the pET32a expression vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The recombinant protein was unique in forming a large fraction of the soluble recombinant protein in E. coli. The antigenicity and immunogenicity of the purified DⅢprotein was tested by Western Blot analysis .The purified domain III protein was emulsified in Freund’s adjuvant (FA) for immunization in mice and rabbits. After 3 times immunized,Rabbits’serum generated 1:4×105 anti-JEV antibody titers .Mice’serum generated 1:7×104 anti-JEV antibody titers and1:2×103 anti-YFV antibody titers .Then resulted in eliciting neutralizing antibodies and protective immunity in ICR mice. These studies can provide useful information for further developing the domain III recombinant protein vaccine against JEV.
引文
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