CAJ | 학술논문

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Westernblot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，Westernblot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CVBDNA疫苗的候选基因，值得进一步深入研究。
목적 제비4충가살기B3병독（CVB3）결구화비결구단백중조질립DNA역묘，병탐토기유도궤체산생체액화세포면역응답적효과。방법 용기인중조기술구건4충CVB3결구화비결구단백중조질립，장각중조질립체외전염진핵세포，용Westernblot검측표체산물；우BALB/c소서후퇴경골전기주사면역，우0、4、8주공면역3차，100μg/차。면역후불동시간검측체액화세포면역응답지표。결과 4충중조질립매절출상응대소적편단，경측서증실위CVB3서렬，Westernblot증실능구재체외진핵세포중표체。pcDNA3/VP2、pcDNA3/VP1、pcDNA3/2A화pcDNA3/3D균가유도소서산생상응적특이성항체、세포독성T림파세포（CTL）화림파세포증식반응、지발형초민반응（DTH），병대치사량적CVB3m、CVB5화CVB2공격구유보호작용，표현위병독공격후제3천혈중병독적도강저，제10천심기병리변화비대조조명현감경，차소서생존솔현저제고。기중이pcDNA/VP1화pcDNA3/3D조보호작용최명현。결론 CVB3결구단백VP1화비결구단백3D질립DNA유가능용작CVBDNA역묘적후선기인，치득진일보심입연구。
Objective To study the humoral and cellular immune responsesinduced 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.