非抗性筛选生长抑素DNA疫苗的构建和免疫效力及安全性研究
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摘要
本研究应用基因克隆、细胞培养、蛋白表达、共聚焦显微观察、酶联免疫测定、RT-PCR、石蜡切片等技术,在生长抑素DNA疫苗pGM-CSF/SS的基础上引入平衡致死系统,构建非抗性筛选生长抑素真核表达质粒pVGS/2SS-asd,并电转化至缺失asd和crp基因的减毒猪霍乱沙门氏菌,将重组菌口服或肌注免疫小鼠后,旨在①探讨其免疫效力及影响因素;②评估其安全性,包括基因整合与毒性分析,为开发安全、高效的促生长DNA疫苗,加快其临床应用奠定基础。
1.非抗性筛选生长抑素真核表达质粒的构建与鉴定
以质粒pGM-CSF/SS为模板扩增生长抑素融合基因片段GS/2SS,之后亚克隆到无抗性真核表达载体pVAX-asd,构建非抗性筛选生长抑素真核表达质粒pVGS/2SS-asd,同时将表达增强型绿色荧光蛋白基因(M4GFP)插入到pVGS/2SS-asd质粒的下游,构建带有绿色荧光蛋白基因的非抗性筛选生长抑素真核表达质粒pGS/2SS-M4GFP-asd。酶切、测序鉴定结果表明,基因的插入位点、方向、序列完全正确。荧光显微观察结果表明,pGS/2SS-M4GFP-asd质粒转染细胞48h后荧光最强烈,且融合蛋白GS/2SS-M4GFP定位于细胞质中。RT-PCR结果表明,pVGS/2SS-asd和pGS/2SS-M4GFP-asd质粒转染细胞48h后均能检测到转录产物。ELISA结果显示,重组质粒表达的融合蛋白均具有SS免疫学活性,表明构建的2种质粒均可在哺乳动物细胞中表达具有生长抑素免疫学活性的融合蛋白。
2.非抗性筛选生长抑素DNA疫苗体外和体内的稳定性
将上述构建的质粒pVGS/2SS-asd电转化至缺失asd基因的减毒猪霍乱沙门氏菌C500,获得非抗性筛选生长抑素DNA疫苗C500(pVGS/2SS-asd)。将重组菌体外传代培养50次后,质粒酶切鉴定结果显示,质粒在传代过程中没有丢失。将重组菌口服和肌注接种小鼠,免疫后2d在肺、脾、肝组织中可检测到重组菌,且重组菌中均可扩增出目的质粒,免疫后14d则检测不到重组菌。由此可见,以减毒沙门氏菌为载体的非抗性筛选生长抑素DNA疫苗在体内和体外均具有良好的稳定性。
3.非抗性筛选生长抑素DNA疫苗的免疫应答及其影响因素
为优化非抗性筛选生长抑素DNA疫苗的免疫程序,本研究设计三个免疫剂量(0.2×10~(10) CFU/只,0.2×10~9 CFU/只,0.2×10~8 CFU/只)、两种免疫次数(1次,2次)、两种免疫间隔(4w,6w)口服免疫昆明雌鼠。ELISA结果显示,试验组小鼠均可以检测到抗SS抗体,且随免疫时间的延长,呈现升高的趋势。总体比较而言,高剂量免疫一次组取得了较高的抗体水平。小鼠体增重结果显示,高剂量免疫一次组取得了相对较好的增重效果。
为探讨非抗性筛选生长抑素DNA疫苗的免疫应答类型,将重组菌C500(pvGS/2SS-asd)口服和肌注免疫Balb/C雌鼠,免疫剂量为0.2×10~(10) CFU/只。采用间接ELISA法检测血浆IgG、IgGl、IgG2a、IgA抗体水平。结果显示,口服免疫和肌注免疫均能诱导产生IgG、IgGl、IgG2a、IgA抗体,且肌注免疫诱导的抗体水平要优于口服免疫。从抗体亚型来看,不论是口服免疫还是肌注免疫产生的IgG1抗体水平均高于IgG2a,表明免疫后均可同时激发Th1型和Th2型免疫应答,但更倾向于Th2型的体液免疫应答。
4.非抗性筛选生长抑素DNA疫苗在小鼠体内的表达分布
将Balb/C雌鼠口服和肌注免疫重组菌C500(pVGS/2SS-asd),剂量为0.2×10~(10)CFU/只,分别于免疫后2d,5d,10d,20d,30d,60d,每组取4只小鼠(含1只对照)无菌采集脑、心、肝、脾、肺、肾、肠、肌肉、卵巢组织,采用RT-PCR方法检测质粒在组织内的表达时相及分布。结果显示,口服免疫后2d可以在心、肝、脾、肺、肾、肠组织中检测到目的基因,第5d时除了卵巢和脑组织之外,其它组织均可扩增到目的基因,第10d仅在肝脏中检测到靶基因。肌注免疫后2d可以在心、肝、脾、肺、肾、肌肉组织中检测到目的基因,与口服组相似的是第5d时除了卵巢和脑组织之外,其它组织均可扩增到目的条带,第10d时除了肝脏、肌肉和脾脏,其它组织均检测不到靶基因。免疫后20d两个免疫组的各个器官均检测不到目的质粒。此研究结果提示非抗性筛选生长抑素DNA疫苗在小鼠体内持续表达时间较短,且有短暂的较高水平表达之后较快被降解。
5.非抗性筛选生长抑素DNA疫苗对小鼠的毒性研究
将昆明小鼠口服和肌注免疫重组菌C500(pVGS/2SS-asd),剂量为0.4×10~(10) CFU/只、0.4×10~(11) CFU/只、0.4×10~(12) CFU/只。免疫后4h、24h、1w、6w采血送检血常规。免疫后1w和6w采集小鼠的心、肝、脾、肺、肾、肠、脑、卵巢(睾丸)、肌肉组织,制备石蜡切片,观察组织是否发生病理变化。血常规检测结果显示,口服和肌注DNA疫苗后,未发现试验组和空菌对照组之间有显著性差异(p＞0.05),石蜡切片结果显示,各个组织器官均正常,提示该疫苗没有引起小鼠毒性反应。
将第3部分的试验末期Balb/C雌鼠处死后,采集心、肝、脾、肺、肾、肠、卵巢、脑、肌肉组织,提取基因组,PCR法检测质粒DNA的染色体整合情况,结果表明,在PCR的最大灵敏度(10拷贝/μg基因组)范围之内,没有发现质粒整合至基因组,提示如果发生整合突变,那么发生频率低于自发突变至少两个数量级。
A series of techniques were used in this study, such as gene cloning, cell culture, protein expression in vitro, Confocal microscopy, ELISA, RT-PCR and paraffin section. In order to construct somatostatin eukaryotic expression plasmid pVGS/2SS-asd without antibiotic resistance gene, a balanced lethal system was introduced into somatostatin DNA vaccine pGM-CSF/SS. Then, the plasmid pVGS/2SS-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain (with double deletion of crp and asd genes) by electroporation, and the recombinant strain C500 (pVGS/2SS-asd) was orally and intra-muscularly immunized against mice. The immunogenicity and safety assessment were discussed here to develop gene immunization techniques to promote growth of animals and to accelerate clinical application of SS gene vaccines.
1. Construction and identification of somatostatin eukaryotic expression plasmid without antibiotic resistance gene
The GS/2SS fusion gene amplified from pGM-CSF/SS plasmid was cloned into pVAX-asd vector without antibiotic resistance gene to obtain plasmid pVGS/2SS-asd. Subsequently, the M4GFP gene was then fused into 3' end of GS/2SS gene in the proper reading frame to construct plasmid pGS/2SS-M4GFP-asd harboring enhanced green fluorescent protein (M4GFP) sequence. The insertion site, direction and sequence of the genes were identified to be correct by restriction endonuclease digestion and sequencing. The Confocal microscopy result showed that the green fluorescence was detected after 48 h of transfection and fusion protein GS/2SS-M4GFP was observed in the cytoplasm. RT-PCR result showed that the GS/2SS somatostatin gene could be detected. ELISA analysis indicated that all the fusion proteins possessed the immunologic competence of SS. This result indicated that the novel constructed plasmids could produce the fusion protein possessing the immunologic competence of SS in mammal cells.
2. The stability analysis of recombinant strain C500 (pVGS/2SS-asd) in vitro and vivo
The plasmid pVGS/2SS-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain by electroporation, and formed SS DNA vaccine C500 (pVGS /2SS-asd) . Strains were passaged 50 generation in vitro, and the results showed plasmid pVGS/2SS-asd was stable in vitro culture. To investigate the bacterial colonization in organs and plasmid stability in vivo, mice were orally and intra-muscularly immunized with C500 (pVGS/2SS-asd). Results showed the strains could be detected in lung, spleen, liver after 2d of immunization. There was no strain to detect at day of 14. The colony PCR showed the target fragment could be amplified from strains. These results showed plasmid pVGS/2SS-asd was stable in vitro and vivo.
3. Immune response and affecting factors of SS DNA vaccine
To optimize the immunization schedule of SS DNA vaccine, three different dose (0.2×10~(10) CFU per mouse, 0.2×10~9 CFU per mouse, 0.2×10~8 CFU per mouse) and booster (0,1) and interval (4w, 6w) were performed in the current study. Results of ELISA showed that the anti-SS-antibodies were detected in the experimental groups, and the titers of antibodies presented a tendency of increase along with the extension of immunization. The high dose with no booster group was received the high antibodies compared with other groups. For the mean body weight of mice, the high dose with no booster group was also obtained the better body gain than the other groups.
To investigate the types of immune response, the Balb/C female mice were orally and intra-muscularly immunized with C500 (pVGS/2SS-asd) , the dose of all groups was 0.2×10~(10) CFU per mouse. ELISA method was used to detect the titer of IgG, IgA and IgG subtype. As a result, the experiment groups could induce IgG, IgG1, IgG2a and IgA antibodies, and the intra-muscular group was better than oral group. For the detection of IgG subtype, the titer of IgG1 was higher than that of IgG2a both in oral and intra-muscular groups. These results indicating that immunization of C500 (pVAX-asd) by oral or intra-muscular administration can induce both Th1 type and Th2 type response, and Th2 type response was better than Th1 type.
4. Expression and distribution pattern of pVGS/2SS-asd in mice various tissues
The Balb/C female mice were orally and intra-muscularly immunized with C500 (pVGS/2SS-asd), the dose of all groups was 0.2×10~(10) CFU per mouse. Mice were killed at 2d, 5d, 10d, 20d, 30d and 60d after immunization and every time 4 mice were sacrificed to collect tissues and organs including brain, heart, liver, spleen, lung, kidney, intestine, muscle and ovary. RT-PCR method was applied to detect the distribution of GS/2SS gene in transcription level. As a result, the target fragment of GS/2SS was detected in the tissues of heart, liver, spleen, lung, kidney and intestine after 2d of oral immunization, respectively. The total tissues were detected the GS/2SS gene with the exceptions of brain and ovary after 5d of oral immunization. At 10d after oral immunization, the GS/2SS gene was detected only in liver tissue. For intra-muscular immunization, the tissues of heart, liver, spleen, lung, kidney and muscular were observed objective fragment at 2d after injection, the result was the same as oral immunization at 5d after immunization. At 10d after immunization, the GS/2SS gene was detected in liver, muscular and spleen tissue. There was no band detected in tissues including oral route and intra-muscular route after 20d of immunization. These results indicated that the expression and distribution pattern of the fusion protein was transiently, and the fusion protein could degrade rapidly.
5. Toxicity assessment of SS DNA vaccine in mice
Kunming mice were orally and intra-muscularly immunized with recombinant vaccine C500 (pVGS/2SS-asd) by the dose of 0.4×10~(10) CFU、0.4×10~(11) CFU、0.4×10~(12) CFU. Blood was collected at 4h、24h、1week、6week after immunization to detect hematology. Tissues such as heart, liver, spleen, lung, kidney, intestine, muscle, ovary or testis, brain were obtained to perform the paraffin sections. The hematology determination was detected with whole blood of mice and there was no significant difference among all groups. The results of paraffin sections showed the vaccine strain was no toxicity.
At the termination of Balb/C mice (part 3), the mice were killed to collect the tissues of heart, liver, spleen, lung, kidney, intestine, muscle, ovary, brain. The genomic DNA was extracted, and then the genomic DNA samples were analyzed by sensitive PCR method. There was no evidence of integration to sensitivity of about 10 copy/μg DNA. If integration occurred at all, the frequency would be at least two orders of magnitude below the spontaneous mutation rate.
1.非抗性筛选生长抑素真核表达质粒的构建与鉴定
以质粒pGM-CSF/SS为模板扩增生长抑素融合基因片段GS/2SS,之后亚克隆到无抗性真核表达载体pVAX-asd,构建非抗性筛选生长抑素真核表达质粒pVGS/2SS-asd,同时将表达增强型绿色荧光蛋白基因(M4GFP)插入到pVGS/2SS-asd质粒的下游,构建带有绿色荧光蛋白基因的非抗性筛选生长抑素真核表达质粒pGS/2SS-M4GFP-asd。酶切、测序鉴定结果表明,基因的插入位点、方向、序列完全正确。荧光显微观察结果表明,pGS/2SS-M4GFP-asd质粒转染细胞48h后荧光最强烈,且融合蛋白GS/2SS-M4GFP定位于细胞质中。RT-PCR结果表明,pVGS/2SS-asd和pGS/2SS-M4GFP-asd质粒转染细胞48h后均能检测到转录产物。ELISA结果显示,重组质粒表达的融合蛋白均具有SS免疫学活性,表明构建的2种质粒均可在哺乳动物细胞中表达具有生长抑素免疫学活性的融合蛋白。
2.非抗性筛选生长抑素DNA疫苗体外和体内的稳定性
将上述构建的质粒pVGS/2SS-asd电转化至缺失asd基因的减毒猪霍乱沙门氏菌C500,获得非抗性筛选生长抑素DNA疫苗C500(pVGS/2SS-asd)。将重组菌体外传代培养50次后,质粒酶切鉴定结果显示,质粒在传代过程中没有丢失。将重组菌口服和肌注接种小鼠,免疫后2d在肺、脾、肝组织中可检测到重组菌,且重组菌中均可扩增出目的质粒,免疫后14d则检测不到重组菌。由此可见,以减毒沙门氏菌为载体的非抗性筛选生长抑素DNA疫苗在体内和体外均具有良好的稳定性。
3.非抗性筛选生长抑素DNA疫苗的免疫应答及其影响因素
为优化非抗性筛选生长抑素DNA疫苗的免疫程序,本研究设计三个免疫剂量(0.2×10~(10) CFU/只,0.2×10~9 CFU/只,0.2×10~8 CFU/只)、两种免疫次数(1次,2次)、两种免疫间隔(4w,6w)口服免疫昆明雌鼠。ELISA结果显示,试验组小鼠均可以检测到抗SS抗体,且随免疫时间的延长,呈现升高的趋势。总体比较而言,高剂量免疫一次组取得了较高的抗体水平。小鼠体增重结果显示,高剂量免疫一次组取得了相对较好的增重效果。
为探讨非抗性筛选生长抑素DNA疫苗的免疫应答类型,将重组菌C500(pvGS/2SS-asd)口服和肌注免疫Balb/C雌鼠,免疫剂量为0.2×10~(10) CFU/只。采用间接ELISA法检测血浆IgG、IgGl、IgG2a、IgA抗体水平。结果显示,口服免疫和肌注免疫均能诱导产生IgG、IgGl、IgG2a、IgA抗体,且肌注免疫诱导的抗体水平要优于口服免疫。从抗体亚型来看,不论是口服免疫还是肌注免疫产生的IgG1抗体水平均高于IgG2a,表明免疫后均可同时激发Th1型和Th2型免疫应答,但更倾向于Th2型的体液免疫应答。
4.非抗性筛选生长抑素DNA疫苗在小鼠体内的表达分布
将Balb/C雌鼠口服和肌注免疫重组菌C500(pVGS/2SS-asd),剂量为0.2×10~(10)CFU/只,分别于免疫后2d,5d,10d,20d,30d,60d,每组取4只小鼠(含1只对照)无菌采集脑、心、肝、脾、肺、肾、肠、肌肉、卵巢组织,采用RT-PCR方法检测质粒在组织内的表达时相及分布。结果显示,口服免疫后2d可以在心、肝、脾、肺、肾、肠组织中检测到目的基因,第5d时除了卵巢和脑组织之外,其它组织均可扩增到目的基因,第10d仅在肝脏中检测到靶基因。肌注免疫后2d可以在心、肝、脾、肺、肾、肌肉组织中检测到目的基因,与口服组相似的是第5d时除了卵巢和脑组织之外,其它组织均可扩增到目的条带,第10d时除了肝脏、肌肉和脾脏,其它组织均检测不到靶基因。免疫后20d两个免疫组的各个器官均检测不到目的质粒。此研究结果提示非抗性筛选生长抑素DNA疫苗在小鼠体内持续表达时间较短,且有短暂的较高水平表达之后较快被降解。
5.非抗性筛选生长抑素DNA疫苗对小鼠的毒性研究
将昆明小鼠口服和肌注免疫重组菌C500(pVGS/2SS-asd),剂量为0.4×10~(10) CFU/只、0.4×10~(11) CFU/只、0.4×10~(12) CFU/只。免疫后4h、24h、1w、6w采血送检血常规。免疫后1w和6w采集小鼠的心、肝、脾、肺、肾、肠、脑、卵巢(睾丸)、肌肉组织,制备石蜡切片,观察组织是否发生病理变化。血常规检测结果显示,口服和肌注DNA疫苗后,未发现试验组和空菌对照组之间有显著性差异(p＞0.05),石蜡切片结果显示,各个组织器官均正常,提示该疫苗没有引起小鼠毒性反应。
将第3部分的试验末期Balb/C雌鼠处死后,采集心、肝、脾、肺、肾、肠、卵巢、脑、肌肉组织,提取基因组,PCR法检测质粒DNA的染色体整合情况,结果表明,在PCR的最大灵敏度(10拷贝/μg基因组)范围之内,没有发现质粒整合至基因组,提示如果发生整合突变,那么发生频率低于自发突变至少两个数量级。
A series of techniques were used in this study, such as gene cloning, cell culture, protein expression in vitro, Confocal microscopy, ELISA, RT-PCR and paraffin section. In order to construct somatostatin eukaryotic expression plasmid pVGS/2SS-asd without antibiotic resistance gene, a balanced lethal system was introduced into somatostatin DNA vaccine pGM-CSF/SS. Then, the plasmid pVGS/2SS-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain (with double deletion of crp and asd genes) by electroporation, and the recombinant strain C500 (pVGS/2SS-asd) was orally and intra-muscularly immunized against mice. The immunogenicity and safety assessment were discussed here to develop gene immunization techniques to promote growth of animals and to accelerate clinical application of SS gene vaccines.
1. Construction and identification of somatostatin eukaryotic expression plasmid without antibiotic resistance gene
The GS/2SS fusion gene amplified from pGM-CSF/SS plasmid was cloned into pVAX-asd vector without antibiotic resistance gene to obtain plasmid pVGS/2SS-asd. Subsequently, the M4GFP gene was then fused into 3' end of GS/2SS gene in the proper reading frame to construct plasmid pGS/2SS-M4GFP-asd harboring enhanced green fluorescent protein (M4GFP) sequence. The insertion site, direction and sequence of the genes were identified to be correct by restriction endonuclease digestion and sequencing. The Confocal microscopy result showed that the green fluorescence was detected after 48 h of transfection and fusion protein GS/2SS-M4GFP was observed in the cytoplasm. RT-PCR result showed that the GS/2SS somatostatin gene could be detected. ELISA analysis indicated that all the fusion proteins possessed the immunologic competence of SS. This result indicated that the novel constructed plasmids could produce the fusion protein possessing the immunologic competence of SS in mammal cells.
2. The stability analysis of recombinant strain C500 (pVGS/2SS-asd) in vitro and vivo
The plasmid pVGS/2SS-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain by electroporation, and formed SS DNA vaccine C500 (pVGS /2SS-asd) . Strains were passaged 50 generation in vitro, and the results showed plasmid pVGS/2SS-asd was stable in vitro culture. To investigate the bacterial colonization in organs and plasmid stability in vivo, mice were orally and intra-muscularly immunized with C500 (pVGS/2SS-asd). Results showed the strains could be detected in lung, spleen, liver after 2d of immunization. There was no strain to detect at day of 14. The colony PCR showed the target fragment could be amplified from strains. These results showed plasmid pVGS/2SS-asd was stable in vitro and vivo.
3. Immune response and affecting factors of SS DNA vaccine
To optimize the immunization schedule of SS DNA vaccine, three different dose (0.2×10~(10) CFU per mouse, 0.2×10~9 CFU per mouse, 0.2×10~8 CFU per mouse) and booster (0,1) and interval (4w, 6w) were performed in the current study. Results of ELISA showed that the anti-SS-antibodies were detected in the experimental groups, and the titers of antibodies presented a tendency of increase along with the extension of immunization. The high dose with no booster group was received the high antibodies compared with other groups. For the mean body weight of mice, the high dose with no booster group was also obtained the better body gain than the other groups.
To investigate the types of immune response, the Balb/C female mice were orally and intra-muscularly immunized with C500 (pVGS/2SS-asd) , the dose of all groups was 0.2×10~(10) CFU per mouse. ELISA method was used to detect the titer of IgG, IgA and IgG subtype. As a result, the experiment groups could induce IgG, IgG1, IgG2a and IgA antibodies, and the intra-muscular group was better than oral group. For the detection of IgG subtype, the titer of IgG1 was higher than that of IgG2a both in oral and intra-muscular groups. These results indicating that immunization of C500 (pVAX-asd) by oral or intra-muscular administration can induce both Th1 type and Th2 type response, and Th2 type response was better than Th1 type.
4. Expression and distribution pattern of pVGS/2SS-asd in mice various tissues
The Balb/C female mice were orally and intra-muscularly immunized with C500 (pVGS/2SS-asd), the dose of all groups was 0.2×10~(10) CFU per mouse. Mice were killed at 2d, 5d, 10d, 20d, 30d and 60d after immunization and every time 4 mice were sacrificed to collect tissues and organs including brain, heart, liver, spleen, lung, kidney, intestine, muscle and ovary. RT-PCR method was applied to detect the distribution of GS/2SS gene in transcription level. As a result, the target fragment of GS/2SS was detected in the tissues of heart, liver, spleen, lung, kidney and intestine after 2d of oral immunization, respectively. The total tissues were detected the GS/2SS gene with the exceptions of brain and ovary after 5d of oral immunization. At 10d after oral immunization, the GS/2SS gene was detected only in liver tissue. For intra-muscular immunization, the tissues of heart, liver, spleen, lung, kidney and muscular were observed objective fragment at 2d after injection, the result was the same as oral immunization at 5d after immunization. At 10d after immunization, the GS/2SS gene was detected in liver, muscular and spleen tissue. There was no band detected in tissues including oral route and intra-muscular route after 20d of immunization. These results indicated that the expression and distribution pattern of the fusion protein was transiently, and the fusion protein could degrade rapidly.
5. Toxicity assessment of SS DNA vaccine in mice
Kunming mice were orally and intra-muscularly immunized with recombinant vaccine C500 (pVGS/2SS-asd) by the dose of 0.4×10~(10) CFU、0.4×10~(11) CFU、0.4×10~(12) CFU. Blood was collected at 4h、24h、1week、6week after immunization to detect hematology. Tissues such as heart, liver, spleen, lung, kidney, intestine, muscle, ovary or testis, brain were obtained to perform the paraffin sections. The hematology determination was detected with whole blood of mice and there was no significant difference among all groups. The results of paraffin sections showed the vaccine strain was no toxicity.
At the termination of Balb/C mice (part 3), the mice were killed to collect the tissues of heart, liver, spleen, lung, kidney, intestine, muscle, ovary, brain. The genomic DNA was extracted, and then the genomic DNA samples were analyzed by sensitive PCR method. There was no evidence of integration to sensitivity of about 10 copy/μg DNA. If integration occurred at all, the frequency would be at least two orders of magnitude below the spontaneous mutation rate.
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