HBV囊膜蛋白与HCV核心蛋白基因联合免疫研究
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摘要
基因免疫既能诱导机体产生体液免疫又能诱导产生细胞免疫，给慢性持续性病毒感染的防治带来了新希望。HCV C蛋白结构保守，诱导产生的免疫应答能够使HCV感染者表现为自限型或非临床型感染，是制备HCV预防及治疗性疫苗较有价值的抗原蛋白。研究HCV C蛋白基因修饰后，所诱导的基因免疫应答和细胞因子对HCV C蛋白基因免疫的调节作用，将有助于HCV疫苗的研制。HBV preS_2S蛋白含有丰富的T、B细胞抗原表位，可诱导产生广泛的抗HBV的免疫应答。本研究构建双表达单元载体Bicistronic，并将HCV C蛋白基因和HBV preS_2S蛋白基因或GM-CSF基因插入其中，制备双价基因免疫质粒，探索了双价基因疫苗免疫应答的规律以及GM-CSF对HCV C基因免疫的调节作用。
一、pcDNA3.0BA Bicistronic载体的构建
以pcDNA3.0为母载体先构建出MCS上酶切点不同的pcDNA3.0A和pcDNA3.0B。然后再组装成具有双CMV启动子的Bicistronic载体pcDNA3.0BA。
二、分泌型信号肽和跨膜蛋白基因修饰HCV C蛋白基因后对基因免疫应答的影响
化学合成HBVpreC基因，分别与HCV C69、C154和C191蛋白基因片段连接，其中pc69 C末端再连接上流感病毒跨膜蛋白TM基因，构建一系列真核表达载体。在Cos-7细胞中进行体外瞬时表达，经~(35)S代谢标记、免疫沉淀检测证实preC可将HCV C蛋白的C69和C154导出细胞外，对C191分泌作
DNA immunization has recently been proven to induce both humoral and cellular immune responses against a range of viruses, so it shows a promising approach to prevention and cure of persistent viral infection. HCV core is highly conserved among HCV genotypes, and its immunogenicity can clear viruses from HCV patients. Although the DNA immunization of HCV core can only induces very weak immune responses, it can be improved by the secretable signal peptide、 transmembrane protein and cytokines.
In this study, bicistronic vector pcDNA3.0BA was constructed . HCV core and HBV preS2S gene or GM-CSF gene were inserted into this vector,and the resulted plasmids were used for transient expression in COS-7 cells in vitro and immunizing mice in vivo.The immune responses to both HBs and HCV,as well as the modulating effects resulting from the co-expression of GM-CSF, were studied.
1. Construction of pcDNA3.0BA bicistrinic
Bicistronic plasmid vector pcDNA3.0BA containing two CMV promoters and two different MCS was constructed from pcDNA3.0. This bicistronic vector can co-express two kinds of genes,with the first gene serving as immunogen and the second gene serving as additional immunogen or as modulator for the immune responses.
2. Secreted signal peptide and transmembrane protein enhance the DNA immunization to HCV core.
HBV preC was fused with HCV C69,C154 and C191 respectively ,and pc69 was further fused with TM from transmembrane protein of influenza virus. A series of eukaryotic expressing plasmids with these chimeric genes were constructed. The results of transient expression in Cos-7 cells labed by 35S Met and immunoprecipation indicated that preC signal peptide can lead C69 and C154 secret into supernatant, but has little effect on C191 ,and pc69TM protein was primarily
一、pcDNA3.0BA Bicistronic载体的构建
以pcDNA3.0为母载体先构建出MCS上酶切点不同的pcDNA3.0A和pcDNA3.0B。然后再组装成具有双CMV启动子的Bicistronic载体pcDNA3.0BA。
二、分泌型信号肽和跨膜蛋白基因修饰HCV C蛋白基因后对基因免疫应答的影响
化学合成HBVpreC基因，分别与HCV C69、C154和C191蛋白基因片段连接，其中pc69 C末端再连接上流感病毒跨膜蛋白TM基因，构建一系列真核表达载体。在Cos-7细胞中进行体外瞬时表达，经~(35)S代谢标记、免疫沉淀检测证实preC可将HCV C蛋白的C69和C154导出细胞外，对C191分泌作
DNA immunization has recently been proven to induce both humoral and cellular immune responses against a range of viruses, so it shows a promising approach to prevention and cure of persistent viral infection. HCV core is highly conserved among HCV genotypes, and its immunogenicity can clear viruses from HCV patients. Although the DNA immunization of HCV core can only induces very weak immune responses, it can be improved by the secretable signal peptide、 transmembrane protein and cytokines.
In this study, bicistronic vector pcDNA3.0BA was constructed . HCV core and HBV preS2S gene or GM-CSF gene were inserted into this vector,and the resulted plasmids were used for transient expression in COS-7 cells in vitro and immunizing mice in vivo.The immune responses to both HBs and HCV,as well as the modulating effects resulting from the co-expression of GM-CSF, were studied.
1. Construction of pcDNA3.0BA bicistrinic
Bicistronic plasmid vector pcDNA3.0BA containing two CMV promoters and two different MCS was constructed from pcDNA3.0. This bicistronic vector can co-express two kinds of genes,with the first gene serving as immunogen and the second gene serving as additional immunogen or as modulator for the immune responses.
2. Secreted signal peptide and transmembrane protein enhance the DNA immunization to HCV core.
HBV preC was fused with HCV C69,C154 and C191 respectively ,and pc69 was further fused with TM from transmembrane protein of influenza virus. A series of eukaryotic expressing plasmids with these chimeric genes were constructed. The results of transient expression in Cos-7 cells labed by 35S Met and immunoprecipation indicated that preC signal peptide can lead C69 and C154 secret into supernatant, but has little effect on C191 ,and pc69TM protein was primarily
引文
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