表达大肠杆菌K99重组干酪乳杆菌口服免疫小鼠的免疫保护性研究

Protection of mice immunized orally with recombinant Lactobacillus casei against E.coli K99

利用干酪乳杆菌(Lactobacillus casei)作为抗原传递系统来刺激机体产生黏膜免疫反应,从而研制有效的黏膜疫苗预防产肠毒素性大肠杆菌(ETEC)的感染。用PCR方法扩增ETECK99基因,克隆到L.casei细胞表面表达载体pLA中,构建了重组表达载体pLA-K99,并将其电转化至L.casei中,在MRS培养基中培养后,经SDS-PAGE、western blot检测目的蛋白的表达,间接免疫荧光分析及流式细胞术检测外源蛋白展示到菌体表面。将重组菌及空质粒菌株分别口服接种SPF级BALB/c小鼠。采集血液样品测定小鼠产生抗K99的特异性IgG,收集小鼠肺部、肠道、阴道冲洗液及粪便样品测定小鼠产生的抗K99的特异性sIgA,并对小鼠进行攻毒保护性试验,免疫组保护率在83%以上,对照组则全部死亡。

The use of live Lactobacillus casei （L .case1） as an antigen delivery vector has been shown to elicit mucosal immu- nity and thus represents a promising strategy for mucosal vaccination against Enterotoxigenic Escherichia coil （ETEC） K99 infec- tion. In this work, the K99 gene was cloned into the expression vector pLA and transformed into the competent cells L. case/. The recombinant protein was detected by SDS-PAGE, western blot. Surface localization of the fusion protein was verified by immunofluo- rescence microscopy and flow cytometry. The recombinant strain harboring pLA- K99/L. casei and pLA/L, casei was orally admin- istered to SPF BALB/c mice. Specific anti-K99 IgG was produced in the serum and secret immunoglobulin A （slgA）. Antibodies were detected in the lung, intestines, vigna fluid and feces by indirect ELISA after oral immunization. More than 83 % immunized mice protected from challenge with C83912 （2×10^3 LD50）.