摘要
目的 构建pQE31/p35原核细胞表达载体 ,研究穿透支原体 (Mpe)相对分子质量 (Mr)为 35× 10 3 蛋白 (P35 )诱生巨噬细胞 (MΦ)分泌TNF α、IL 1β、IL 6等细胞因子 (CKs)的作用。方法 PCR扩增p35全长 10 0 2bp基因片段 ,克隆至pQE31原核细胞表达载体 ,用PCR介导的突变将p35基因中两个“TGA”终止密码子定点突变为“TGG” ,使其在大肠杆菌 (E .coli)中编码表达色氨酸。IPTG诱导pQE31/p35在E .coli中表达目的蛋白rP35 ,用Ni NTASpin亲和纯化 ,Westernblot鉴定表达产物。用酶联免疫吸附试验 (ELISA)检测P35刺激小鼠MΦ分泌TNF α、IL 1β和IL 6的量。结果 PCR扩增出约 1kb的DNA片段 ,克隆至pQE31载体 ,筛选到阳性克隆pQE31/p35 ,p35基因中两个“TGA”终止密码子成功突变成“TGG”。IPTG诱导pQE31/p35在E .coli中表达出Mr 约 37× 10 3 的蛋白质 ,Westernblot鉴定其为rP35。ELISA结果表明 ,P35能刺激小鼠MΦ分泌大量的TNF α、IL 1β和IL 6。结论 P35能诱导MΦ分泌TNF α、IL 1β、IL
Objective To study the effect of Mycoplasma pene tr ans M r 35×103 protein (P35) in inducing tumor necrosis factor α(TNF-α) , interleukin-1β(IL-1β) and interleukin-6(IL-6) secreted by murine macroph ages. Prokaryotic expression vector pQE31/p35 was constructed and recombinant fu sion protein P35(rP35) was expressed in E.coli. Methods The p35 gene, amplified by polymerase chain reaction(PCR), was cloned into pQ E31. PCR-mediated mutagenesis was used to change the two “TGA” triplets to “ TGG” triplets within the p35 gene. Production of recombinant protein was induce d by the addition of IPTG, rP35 was purified with a Ni-NTA Spin Kit and rP35 pu rification was analyzed by Western blot. TNF-α, IL-1β, IL-6 secreted by mur ine macrophages were measured by ELISA. Results About 1kb PCR amplification was cloned into pQE31 and two “TGA” triplets within the p35 gene were successfully changed to “TGG” triplets encoding tryptophan in E.c oli. The pQE31/p35 expressed a protein with a calculated molecular mass of 37 ×103 in E.coli. Western blot showed that the M r 37×103 protein w as rP35. P35 stimulated murine macrophages to produce proinflamatory cytokines i ncluding TNF-α, IL-1β and IL-6. Conclusion P3 5 can induce secretion of TNF-α, IL-1β and IL-6 by murine macrophages. [
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第9期707-710,共4页
Chinese Journal of Microbiology and Immunology
基金
湖南省自然科学基金资助课题 (0 2JJY2 0 2 5 )
湖南省卫生厅资助课题 (Y0 2 0 66)
