柯萨奇B3病毒结构和非结构蛋白DNA 疫苗诱导小鼠产生免疫应答的研究

目的 制备4种柯萨奇B3病毒（CVB3）结构和非结构蛋白重组质粒DNA疫苗，并探讨其诱导机体产生体液和细胞免疫应答的效果。方法 用基因重组技术构建4种CVB3结构和非结构蛋白重组质粒，将各重组质粒体外转染真核细胞，用Western blot检测表达产物；于BALB／c小鼠后腿胫骨前肌注射免疫，于0、4、8周共免疫3次，100μg/次。免疫后不同时间检测体液和细胞免疫应答指标。结果 4种重组质粒酶切出相应大小的片段，经测序证实为CVB3序列，Western blot证实能够在体外真核细胞中表达。pcDNA3/vp2、pcDNA3/VP1、pcDNA3/2A和pcDNA3/3D均可诱导小鼠产生相应的特异性抗体、细胞毒性T淋巴细胞（CTL）和淋巴细胞增殖反应、迟发型超敏反应（DTH），并对致死量的CVB3m、CVB5和CVB2攻击具有保护作用，表现为病毒攻击后第3天血中病毒滴度降低，第10天心肌病理变化比对照组明显减轻，且小鼠生存率显著提高。其中以pcDNA/VP1和pcDNA3／3D组保护作用最明显。结论 CVB3结构蛋白VP1和非结构蛋白3D质粒DNA有可能用作CVB DNA疫苗的候选基因，值得进一步深入研究。

Study on the humoral and cellular immune responses in the mice induced with DNA vaccines of CVB3 structural and nonstructural protein

Objective To study the humoral and cellular immune responses induced by four DNA vaccines of CVB3 structural and nonstructural proteins. Methods VP1, VP2,2A and 3D genes were amplified from CVB3 infected cells with RT PCR and recombinanted into eukaryotic expression plasmid, pcDNA3. The four recombinant plasmids were injected into the tibialis anterior muscle of the mice. The mice were subsequently given booster dose twice at 4 week intervals. In the different times post immunization the humoral and cellular immune responses were detected. Results Four recombinant plasmids were confirmed with restriction digestion and sequencing to contain target gene segments and expressed in COS 7 cells in vitro shown by Western blot. All of four recombinant plasmids induced corresponding specific antibody response, lymphocyte proliferation, CTL, DTH reaction and protected the mice from challenge of the lethal CVB3m, CVB2, CVB5. Of all immunized groups pcDNA3/VP1 and pcDNA3/3D groups induced more stronger immune responses than other groups. Conclusion VP1 and 3D of CVB3 are potential to be used as the candidate genes, and so is valuable to study furthermore.