双歧杆菌肠道内调控表达肠道病毒71型VP1蛋白的实验研究

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。

Oral immunization of mice using Bifidobacterium iongum expressing the VP1 protein from enterovirus 71

Objectives To construct a Bifidobacterium recombinant with the VP1 gene from enterovirus 71 （EV71） and to create an oral vaccine to prevent EV71 infection. Methods A VP1 expression vector （pBBADs VP1） was constructed by amplifying the VP1 gene of EV71 and inserting it into the E. coli-Bifidobacterium shuttle expression vector pBBAD/Xs. Expression of VP1 protein in pBBADs-VPl-transformed bacteria was detected using Western blotting. BALB/ c mice were divided randomly into four groups. The B-VP1 group was orally immunized with VP1 expressing B. longum, the VP1 group was intraperitoneally （i. p. ） administered the recombinant VP1 protein, one control group was orally im- munized with saline （the saline group）, and another control group was orally immunized with GFP-transformed B. lon gum （the GFP group）. The immune response of BALB/c mice orally immunized with pBBADs-VPl-transformed bacteria to protect against EV71 injection was observed. Observed aspects included the virus-neutralizing titer, the level of anti- EV71 VP1 antibodies, the induction of a Thl immune response in the spleen, and Peyefs patches. Results The virusneutralizing titer and level of anti-EV71-VP1 antibodies increased markedly in the B VP1 group and VP1 group compared to the GFP group and saline group. The BVP1 group had a higher virus-neutralizing titer after immunization and 4, 8, and 12 weeks later （1：2, 1：2, 1.. 2, and 1：2, respectively） than did the GFP group and saline group （0 in both） （F 10.19, P〈0.05）. The VP1 group also had a higher virus-neutralizing titer after immunization and 4, 8, and 12 weeks later （1：2, 1：4, 1：4, and 1：2, respectively） than did the GFP group and saline group （0 in both） （F=7.35, P〈0.05）.The VP1 group had higher levels of anti-EV71-VP1 antibodies 4, 8, and 12 weeks after immunization in comparison to theB-VP1 group （0.20~0.06 vs. 0.14±0.05, 0.34±0.07 vs. 0.30±0.05, 0.45±0.10 vs. 0.39±0.09, respectively） （F=5.58, P〈0.05）. A significantly greater Thl cellular immune response was noted in both the B-VP1 group and the VP1 group in comparison to the control groups （the GFP group and saline group） （F--6.87,P〈0.05）. These find ings suggest that VPl-expressing B. longum may readily induce a Thl response pattern in the intestines while the injected VP1 protein facilitates the induction of systemic immunity. A lethal challenge test indicated that newborn rats in the VP1 group and B-VP1 group had a survival rate of 20.00% and 16.67% on day 15 （dose= 1, 000 LD50 ） while none of the mice in the control groups survived. Conclusion These results indicate that a novel oral vaccine featuring B. Zongurn expressing the VP1 protein may successfully elicit a specific immune response against EVT1 infection. Immunizing mouse mothers with recombinant VPl-expressing B. longum can confer protection to newborn mice.