问号钩端螺旋体鞭毛蛋白相关蛋白FliH／I／Y／N基因原核表达和膜定位  

Prokaryotic expression of Leptospira interrogans FIiH/I/Y/N genes and determination of the protein locations in leptospiral envelope

作　　者：徐韩飞[1] 阮萍[3] 廖苏梅[1] 杨平[2] 毛亚飞[1] 李立伟[1] 严杰[1] 

机构地区：[1]浙江大学医学院病原生物学系,杭州310058 [2]浙江大学医学院电镜室,杭州310058 [3]浙江省绍兴文理学院医学院,312000

出　　处：《中华微生物学和免疫学杂志》2008年第7期597-601,共5页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：国家自然科学基金面上项目（30370072）

摘　　要：目的克隆问号钩端螺旋体（简称钩体）鞭毛相关蛋白编码基因fliH、fliI、fliY和fliN并构建其原核表达系统，制备表达产物抗血清并了解其蛋白的定位。方法以苯酚-氯仿法提取的问号钩体黄疸出血群赖型赖株基因组DNA为模板，用PCR扩增全长fliH、fliI、fliY和fliN基因片段，T-A克隆后测序，继而构建上述目的基因克隆的原核表达系统。采用SDS-PAGE和BioRad凝胶图像分析系统检查重组蛋白rFliH、rFliI、rFliY和rFliN的表达情况，Ni-NTA亲和层析法提纯目的表达产物。皮下免疫家兔获得4种目的重组蛋白抗血清，用ELISA和Western blot分别检测抗血清效价并了解抗血清与相应抗原结合的能力。采用免疫电镜技术对fliH、fliI、fliY和fliN进行定位。结果PCR扩增获得大小分别为924、1365、1065和318 bp的全长fliH、fliI、fliY和fliN基因片段，与报道的序列比较，其核苷酸和氨基酸序列相似性均为100％。所构建的原核表达系统均能有效地表达目的重组蛋白，其产量均约为20％。rFliH、rFliI、rFliY和rFliN蛋白免疫家兔后能产生抗体，其兔抗血清ELISA效价达到1：100000以上，并分别识别钩体相应重组蛋白和膜蛋白提取物而出现明显的Western杂交条带。fliH、fliI、fliY和fliN蛋白分布于问号钩体内膜、外膜或内外膜之间。结论本研究成功地构建了能高效表达问号钩体鞭毛相关蛋白fliH、fliI、fliY和fliN的原核表达系统，并获得了能有效识别上述蛋白抗原的高效价抗血清。鞭毛相关蛋白fliH、fliI、fliY和fliN是问号钩体内膜或外膜蛋白成分。Objective To clone flirt, fill, fliY and tiN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the locations of fliH、fliI、fliY and fliN. Methods The fliH, flil, fliY and fliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotic expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression products. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain antisera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of fliH、fliI、fliY and fliN. Results Segments of fliH、fliI、fliY and fliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and putative amino acid sequences from the four genes were 100% compared with the reported sequences. The constructed prokaryotic systems efficiently expressed rFliH, rFliI, rFliY and rFliN with the outputs of approximate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliI, rFliY or rFliN could produce antibody. The antisera had the titers above 1：100 000, and could recognize the corresponding recombinant proteins and membrane proteins of L. interrogans to display positive Western hybridization bands. fliH、fliI、fliY and fliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The prokaryotic expression systems was successfully constructed in this study, which could efficiently express flagellum-associated proteins fliH、fliI、fliY and fliN

关 键 词：问号钩端螺旋体 鞭毛相关蛋白 基因克隆 原核表达 免疫原性/定位 

分 类 号：R514[医药卫生—内科学] R377[医药卫生—临床医学]