钩端螺旋体groEL基因原核表达及其产物免疫保护作用

Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters

目的:构建问号钩端螺旋体(简称钩体)黄疸出血群赖型赖株groEL基因原核重组表达系统,了解重组表达的GroEL蛋白(rGroEL)对金地鼠的免疫保护作用。方法:采用高保真PCR扩增问号钩体赖株groEL基因并测序,常规方法构建groEL基因原核表达系统。采用SDS-PAGE联合Bio-Rad凝胶图像分析系统检查rGroEL表达情况及其可溶性,Ni-NTA亲和层析法提纯rGroEL。观察rGroEL免疫金地鼠对问号钩体赖株感染的保护率。采用MAT法检测免疫动物血清与我国流行问号钩体血清群交叉凝集效价。结果:所克隆的groEL基因核苷酸和氨基酸序列与GenBank中相应基因完全相同。所构建的groEL基因原核表达系统能表达可溶性rGroEL。100和200μgrGroEL对金地鼠的免疫保护率分别为50.0%和75.0%。rGroEL免疫金地鼠血清可不同程度地凝集各问号钩体血清群。结论:GroEL蛋白是问号钩体属特异性保护性抗原,可用于制备通用性钩体基因工程疫苗。

Objective： To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein （rGroEL） in LVG hamsters. Methods： The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroELprotein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L. interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L. interrogans serogroups were detected using MAT. Results： The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200μg rGroEL in LVG hamsters were 50.0% and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L. interrogans serogroups tested with different levels. Conclusion： The GroEL protein is a genus-specific immunoprotective antigen of L. interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.