赖型钩端螺旋体毒力相关蛋白InvA的表达及其促细胞增殖效应  

Expression of virulence-associated protein InvA of the strong virulent L.interrogans serovar Lai and its effect on ANA-1 cell proliferation

作　　者：朱庆平[1] 鲍朗[1] 张会东[1] 赵计林[1] 龙洋[1] 吴悦涵[1] 赵明才[1] 

机构地区：[1]四川大学华西基础医学与法医学院感染免疫研究室,成都610041

出　　处：《中华微生物学和免疫学杂志》2004年第11期866-869,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 3 0 0 70 670 )

摘　　要：目的　在大肠杆菌中表达赖型钩端螺旋体毒力相关蛋白InvA ,并观察该蛋白对ANA 1细胞的增殖作用。方法　将invA基因克隆于pET32a(+)载体 ,在大肠杆菌BL2 1(DE3)中表达带组氨酸标签的Trx InvA融合蛋白 ,经亲和层析获得纯化Trx InvA蛋白。以不同浓度的Trx InvA融合蛋白作用于ANA 1细胞 ,以四氮唑复合物 [2 ,3 bis(2 methoxy 4 nitro sulfophenyl) 5 〔(Phenylamino)carbonyl〕 2H tetrazoiumhydrixide,XTT]法检测细胞增殖的变化 ,分析InvA蛋白的细胞功能。结果　成功构建pETIN VA原核表达载体 ,在大肠杆菌BL2 1(DE3)中表达了相对分子质量 (Mr)约为 35× 10 3的Trx InvA融合蛋白 ,该蛋白能促进ANA 1细胞的增殖。结论　表达并纯化的TrxObjective To express virulence-associated protein InvA from a strong virulent L.interrogans serovar Lai in E. coli BL21(DE3) and to investigate its effects on cell proliferation of ANA-1. Methods The (invA) gene was cloned into prokaryotic expression vector pET32a(+). The recombination expression plasmid (pETINVA) was transformed into E. coli BL21(DE3). The fused protein Trx-InvA which had a 6-His tag was expressed after induction by IPTG and purification. The effect on ANA-1 cell proliferation to different concentrations of Trx-InvA was observed using the XTT assay. Results The recombination plasmid pETINVA was successfully constructed with high level expression of recombination fusion protein Trx-InvA in E. coli BL21(DE3). The fusion protein Trx-InvA was purified by His-affinity column. SDS-PAGE showed that the molecular relative mass(M_r) of Trx-(InvA) was about 35×10~3. The XTT assay indicated that Trx-InvA promoted a concentration-depended cell proliferation of ANA-1 when the concentration of Trx-InvA was less than 0.5μg/ml, but Trx-InvA failed to increase ANA-1 cell proliferation any more at 1.0μg/ml and 2.0μg/ml. Conclusion These results provided an experimental model for identifying pathogenic potential and molecular functions of InvA. [

关 键 词：原核表达载体 细胞增殖 相关蛋白 ANA 钩端螺旋体 融合蛋白 大肠杆菌 纯化 亲和层析 基因克隆 

分 类 号：R377[医药卫生—病原生物学]