丙型肝炎病毒基因疫苗构建物的实验研究

Experimental study of immunization of mice with hepatitis C virus genetic vaccine constructs

目的探讨丙型肝炎病毒（ＨＣＶ）基因疫苗诱发小鼠免疫反应，为进一步开展ＨＣＶ基因疫苗研制奠定基础。方法将编码ＨＣＶⅡ型结构蛋白基因片段插入真核细胞表达载体ｐＣＤＳＲα１中，形成基因疫苗构建物ｐＣＤＨＣＶ１。从股四头肌免疫Ｂａｌｂ／ｃ小鼠，用ＥＬＩＳＡ和３ＨＴｄＲ掺入法检测免疫鼠抗体反应和外周血单个核细胞（ＰＢＭＣ）对ＨＣＶ抗原的增殖反应。结果基因疫苗ｐＣＤＨＣＶ１（１００μｇ／只）３～４次接种小鼠后（ｎ＝１２），血清抗体水平达０．７１±０．０８～０．７７±０．０６（Ａ值，下同），空载体ｐＣＤＳＲα１免疫鼠血清抗体阴性；免疫鼠（ｎ＝８）抗体水平达高峰后（０．７１），持续检测１８周未见下降趋势（０．６８±０．０６～０．７５±０．０７）；ｐＣＤＨＣＶ１免疫小鼠３个月后，用聚合酶链反应（ＰＣＲ）仍可从肌肉组织中扩增出特异性ＨＣＶｃＤＮＡ片段；免疫鼠ＰＢＭＣ对ＨＣＶ合成肽ＣＰ９、基因重组抗原Ｃ、Ｅ１刺激后均出现增殖反应，刺激指数（ＳＩ）分别为４．０７±１．５８、３．８８±０．７０和３．６９±１．３０，与ｐＣＤＳＲα１免疫鼠相比，差异有显著性（Ｐ＜０．００１）。结论构建的ＨＣＶ结构区基因疫苗可诱发Ｂａｌｂ?

Objective To inquire into the immune responses to expression protein in mice immunized with genetic vaccine of hepatitis C virus(HCV) and lay a foundation for HCV genetic vaccine development in future. Methods The gene fragments coding C and most E regions of HCV type wereinserted into pCDSR1 of eukaryotic expression vector and formed genetic vaccine constructs of pCDHCV1 and then was injected into the quadriceps muscles of Balb/c mice. The serum antiHCV level of mice was tested by ELISA and peripheral blood mononuclear cell(PBMC)proliferative responses to HCV antigens were detected by3HTdR incorporation method(cpm). ResultsT5BZThe serum antibody level reached to 0.710.080.770.06 (A value,the same below) after genetic vaccine pCDHCV1(100 g/mouse) were inoculated into the mice(n=12) three or four times while blank vector pCDSR1 could not induce the mice(n=8) to generate antibody response in same way.After the antibody levels in mice(n=8) immunized by pCDHCV1 had ascended to peak value(0.71), there was no trend of descending during the following 18 weeks of detection(0.680.060.750.07). Specific fragment of HCV cDNA identified by polymerase chain reaction (PCR) from DNA extracted from the muscles of the mice after pCDHCV1 had beed inoculated three months. PBMC proliferative responses to HCV synthetic peptides CP9 and gene recombinant antigens CE1 in the mice immunized with pCDHCV1 were detected and its stimulaion indexes(SI) were 4.071.583.880.70 and 3.691.13 respectively and there was a significant difference (P<0001) as compared with that of PBMC in mice immunized with pCDSR1. Conclusion These investigations demonstrated that genetic vaccine constructs made of HCV structural region can induce Balb/c mice to generate antibody and PBMC proliferative responses to HCV antigens via DNA immunization.