霍乱弧菌减毒活疫苗候选株IEM109的构建

A new live cholera vaccine candidate constructed by gene recombination

目的 构建保护性抗原基因整合于染色体上的霍乱弧菌活疫苗候选株 ,减弱毒力和增强其遗传稳定性。方法 以我们筛选的天然不含霍乱毒素的疫苗候选株IEM1 0 1为出发株 ,通过基因重组技术将IEM1 0 1染色体上与噬菌体复制整合有关的TLC因子、attRS位点及具细胞毒作用的RTX基因簇同时进行缺失 ,并插入具免疫原性的ctxB基因及介导噬菌体免疫的rstR基因 ,构建疫苗候选株IEM1 0 9。GM1 ELISA法检测IEM1 0 9中ctxB基因的表达情况。细胞培养观察候选株对哺乳动物细胞Hep 2的毒性作用。结果 PCR及原位杂交均证实霍乱弧菌疫苗候选株IEM1 0 9中TLC因子、attRS位点、rtxA、rtxC基因被缺失 ,而ctxB、rstR基因整合入染色体DNA。GM1 ELISA示ctxB基因表达良好。IEM1 0 9对Hep 2细胞的毒性作用消失。结论 构建了出发株染色体上毒力相关基因簇被保护性抗原基因替换的重组改造菌株 ,可作为候选株进行进一步的免疫效果考核。

Objective To develop an attenuated live cholera candidate with the substitution of protective genes for the virulent elements, to enhance its safety and genetic stability. Methods A chromosomal fragment containing TLC element, attRS of CTXΦ integration site, and RTX cluster responsible for the cytotoxic activity for mammalian cells, were deleted through gene recombination approach from the cholera vaccine prototype strain IEM101. The protective genes of rstR related to CTXФ superinfection exclusion, and toxin subunit gene ctxB were integrated in the same location simultaneously to generate the candidate IEM109. Expression of ctxB was detected with GM1-ELISA, and the cytotoxic effect was evaluated in Hep-2 culture. Results TLC genes, attRS and rtxAC were deleted while ctxB and rstR were integrated in the same site on the IEM101 chromosome DNA. ctxB was expressed and the cytotoxic effect on Hep-2 was negative. Conclusion We constructed a new candidate of our vaccine candidate series.