论文部分内容阅读
目的构建重组肠道病毒71VP1基因双歧杆菌,制成口服疫苗,用于EV71感染预防。方法扩增EV71病毒VP1基因并将其插入到大肠埃希菌-双歧杆菌穿梭表达载体pBBAD/Xs中,构建VP1表达载体(pBBADs-VP1),通过Western blot检测VP1蛋白在pBBADs-VP1转化菌中的表达。选取BALB/c小鼠,分为B-VP1组、VP1组、GFP组和生理盐水组,B-VP1组口服pBBADs-VP1-转化双岐杆菌,VP1组腹腔注射大肠埃希菌表达纯化的重组VP1蛋白,GFP组口服pBBADs-GFP转化双岐杆菌,生理盐水组口服生理盐水,观察pBBADs-VP1-转化双岐杆菌口服免疫对EV71感染的免疫反应:包括病毒的中和抗体滴度、抗EV71-VP1抗体以及诱导脾脏和淋巴集结中Th1免疫反应。结果与GFP组及生理盐水组比较,B-VP1组及VP1组中和抗体滴度及抗EV71-VP1抗体水平,均显著增加;口服表达VP1蛋白长双歧杆菌和注射VP1蛋白都会诱导Th1细胞因子免疫反应(P<0.05),表达VP1蛋白长双歧杆菌较易诱导局部肠道的Th1模式,而注射VP1蛋白较易诱导全身性免疫。攻毒试验显示,VPI和B-VP1组新生鼠攻击后第15d(剂量1 000LD50)存活率分别为20.00%和16.67%,对照组无小鼠存活。结论利用长双歧杆菌表达VP1蛋白的口服疫苗可能成功激发针对EV71病毒感染的特异性免疫应答。用表达VP1的重组双歧杆菌免疫母鼠,可以赋予新生小鼠以保护。
Objective To construct the recombinant enteroviruses 71VP1 gene Bifidobacterium and make oral vaccine for EV71 infection prevention. Methods The VP1 gene of EV71 virus was amplified and inserted into shuttle vector pBBAD / Xs of Escherichia coli-Bifidobacterium. VP1 expression vector (pBBADs-VP1) was constructed. The VP1 protein of pBBADs-VP1 In the expression. BALB / c mice were selected and divided into B-VP1 group, VP1 group, GFP group and saline group. B-VP1 group was orally administered with Bifidobacterium pBBADs-VP1-B12 and VP1 group was orally injected with recombinant E. coli VP1 protein and GFP group, oral administration of pBBADs-GFP to Bifidobacterium was inoculated in normal saline group, and oral immunization with pBBADs-VP1-Bifidobacterium Bifidobacterium was used to detect the immune response to EV71 infection including neutralizing antibody titers of virus and anti-EV71 -VP1 antibody as well as inducing a Th1 immune response in the spleen and lymph nodes. Results Compared with GFP group and NS group, the neutralizing antibody titers and anti-EV71-VP1 antibody levels in B-VP1 group and VP1 group were significantly increased. Oral administration of VP1 Bifidobacterium and VP1 protein induced Th1 cells Factor immune response (P <0.05), the expression of VP1 protein Bifidobacterium more likely to induce local intestinal Th1 mode, while the injection of VP1 protein more likely to induce systemic immunity. Toxicity test showed that the survival rate of mice in VPI and B-VP1 group after 15 days of challenge (dose 1 000 LD50) were 20.00% and 16.67% respectively, and no mice in the control group survived. Conclusions Oral vaccines expressing VP1 protein from B. longum may successfully elicit specific immune responses against EV71 virus infection. Immunization of female mice with recombinant Bifidobacteria expressing VP1 may confer protection to neonatal mice.