致病钩体表层膜蛋白新基因(Lslp)的克隆表达及免疫原性研究  

Cloning,Expression and Immunogenicity of S-layer Membrane Protein Gene(Lslp) of Pathogenic Leptospira interrogans

作　　者：胡昌华[1,2] 鲍朗[3] 谢建平[1] 张会东[3] 

机构地区：[1]西南师范大学生命科学学院现代生物医药研究所 [2]四川大学华西医学中心成都610041 [3]四川大学华西医学中心

出　　处：《微生物学报》2004年第4期500-503,共4页Acta Microbiologica Sinica

基　　金：国家自然科学基金项目 ( 3 0 0 70 670 ) ;重庆市应用基础研究项目 ( 2 0 0 2 7481)~~

摘　　要：克隆表达钩端螺旋体表层膜蛋白新基因Lslp并分析表达产物的免疫原性。根据前期研究得到的致病钩体新基因Lslp(GenBankAF32 5 80 7)的序列设计引物 ,在 6株致病钩体中扩增Lslp基因并测序。以BamHⅠ酶切Lslp和pGEX 1 λT ,构建重组质粒并用酶切和PCR鉴定 ,进一步在大肠杆菌中诱导表达 ,并进行免疫印迹分析 ;纯化表达产物免疫家兔 ,ELISA检测血清抗体滴度。结果显示Lslp在 6株致病钩体中均能扩增出相应片段 ,且序列同源性达到99 6 % ;构建高效原核表达重组质粒pGST LslP ,经IPTG诱导在大肠杆菌中可表达出 6 6kDGST融合蛋白 ,并能与全钩抗血清发生免疫印迹反应 ;将上述融合蛋白免疫新西兰大白兔产生 1 :5 1 2 0高滴度的IgG抗体。研究结果提示致病钩体膜蛋白新基因Lslp可在大肠杆菌进行高效表达 ,表达产物能被全钩抗血清识别 。S layer proteins was assumed to play crucial role in the virulence of pathogens.In order to elucidate of role of Leptospira interrogans S layer protein, the encoding gene was cloned and expressed in E.coli . The immunogenecity of the recombinant protein was assayed. The primer for PCR amplification was based on previous data (GenBank AF325807), genomic DNA from 6 strains of Leptospira interrogans were used as template for amplification separately. The resulting PCR fragments of Lslp genes from 6 different pathogenic strains were sequenced and aligned. Lslp gene of Leptospira interrogans serovar Lai was subjected to further studies. The recombinant plasmid pGST Lslp was constructed and used to transfer the host E.coli JM109 The recombinant fusion protein was purified and used to immunize the rabbit. The antibody titers was determined by ELISA with Lslp immunized rabbit serum. The results showed the new S layer protein gene was highly conserved in 6 strains of pathogenic Leptospira interrogans . The sequence homology was 99 6%. The gene was not detected in nonpathogenic Leptospira. The apparent molecular weight of the expressed GST fusion protein was 66 kD. Western blot demonstrated a clear band reacted with rabbit anti pan Leptospira serum. The maximum antibody titers of Lslp immunized reaction reached 1:5120 These data indicate that the surface layer protein (Lslp) of Leptospira interrogans can be recognized by host and its role in the host pathogen interaction needs further investigation. The study provide clue for further explaining the molecular mechanism of pathogenic Leptospira and screening the target for the vaccine design.

关 键 词：克隆 表达 钩端螺旋体 表层膜蛋白 基因 免疫原性 

分 类 号：R346[医药卫生—基础医学]