肺炎支原体黏附蛋白P1羧基末端的克隆表达及检测

发布时间：2018-08-16 14:36

【摘要】：肺炎支原体(Mycoplasma Pneumoniae,MP)是引起人类非典型肺炎和多种呼吸道感染的病原体之一,人感染后还会引起心肌损害、神经系统损害、肾脏损害、传染性单核细胞增多症、川崎病等并发症。肺炎支原体对青霉素类药物不敏感,只对大环内酯类、四环素类药物敏感。因此,尽早检测出肺炎支原体感染,对有效治疗由其引发的疾病具有重要意义。肺炎支原体能感染人关键是其可以黏附人的呼吸道上皮细胞,而黏附蛋白P1是肺炎支原体的主要黏附蛋白,也是肺炎支原体的主要免疫原,在P1羧基末端含有多个抗原决定簇。本研究以肺炎支原体FH株基因组DNA为模板,利用聚合酶链式反应(PCR)扩增获得黏附蛋白P1基因羧基末端部分编码序列,再将其克隆到克隆载体pEASY-T1上,进而得到了pEASY-MP-P1F这一重组质粒。后续实验对pEASY-MP-P1F进行基因测序检查,把测序结果正确的P1F基因克隆到表达载体pET32a(+)上,构建pET32a-MP-P1FE这一重组表达质粒。接着对pET32a-MP-P1FE中的P1FE进行测序,检测正确后转化大肠杆菌BL21(DE3),利用IPTG诱导表达,以Ni~(2+)亲和层析柱对pET32a-MP-P1FE表达的重组蛋白进行纯化,SDS-PAGE显示纯化的重组蛋白相对分子质量为50kDa至60kDa,与预计值55.9kDa一致。Western Blotting、ELISA免疫检测证实,重组蛋白与感染肺炎支原体病人血清中的抗体有特异性反应。因此,此次研究成功克隆表达了具有免疫原性的黏附蛋白P1羧基末端基因,为进一步研究开发肺炎支原体诊断试剂和疫苗等奠定了良好的基础。
[Abstract]:Mycoplasma pneumoniae MP is one of the pathogens that cause human atypical pneumonia and multiple respiratory tract infections. Human infection can also cause myocardial damage, nervous system damage, kidney damage, infectious mononucleosis, Kawasaki disease and other complications. Mycoplasma pneumoniae is not sensitive to penicillin but only to macrolides and tetracyclines. Therefore, early detection of mycoplasma pneumoniae infection is of great significance for the effective treatment of diseases caused by mycoplasma pneumoniae. Mycoplasma pneumoniae can infect human respiratory epithelial cells, and adhesion protein P1 is the main adhesion protein of mycoplasma pneumoniae and the main immunogen of mycoplasma pneumoniae. There are many antigenic determinants at the end of P1 carboxyl group. In this study, the genomic DNA of Mycoplasma pneumoniae FH strain was used as template, and the partial coding sequence of the carboxyl terminal of adhesion protein P1 gene was obtained by polymerase chain reaction (PCR) amplification. Then it was cloned into the clone vector pEASY-T1 and the recombinant plasmid pEASY-MP-P1F was obtained. The pEASY-MP-P1F gene was sequenced and the P1F gene was cloned into the expression vector pET32a (). The recombinant expression plasmid pET32a-MP-P1FE was constructed. Then the P1FE in pET32a-MP-P1FE was sequenced and transformed into Escherichia coli BL21 (DE3), and the expression was induced by IPTG. The recombinant protein expressed in pET32a-MP-P1FE was purified by Ni ~ (2) affinity chromatography column. SDS-PAGE showed that the molecular weight of the purified recombinant protein was from 50kDa to 60 kDa, which was consistent with the predicted value of 55.9kDa. Western blotting Elisa was used to confirm the molecular weight of the purified recombinant protein. The recombinant protein reacts specifically with antibodies in the sera of patients infected with Mycoplasma pneumoniae. Therefore, this study successfully cloned and expressed the adhesion protein P1 carboxyl terminal gene with immunogenicity, which laid a good foundation for further research and development of mycoplasma pneumoniae diagnostic reagent and vaccine.
【学位授予单位】：湖南科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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