【摘要】：肺炎支原體(Mycoplasma Pneumoniae,MP)是引起人類非典型肺炎和多種呼吸道感染的病原體之一,人感染后還會(huì)引起心肌損害、神經(jīng)系統(tǒng)損害、腎臟損害、傳染性單核細(xì)胞增多癥、川崎病等并發(fā)癥。肺炎支原體對(duì)青霉素類藥物不敏感,只對(duì)大環(huán)內(nèi)酯類、四環(huán)素類藥物敏感。因此,盡早檢測(cè)出肺炎支原體感染,對(duì)有效治療由其引發(fā)的疾病具有重要意義。 肺炎支原體能感染人關(guān)鍵是其可以黏附人的呼吸道上皮細(xì)胞,而黏附蛋白P1是肺炎支原體的主要黏附蛋白,也是肺炎支原體的主要免疫原,在P1羧基末端含有多個(gè)抗原決定簇。 本研究以肺炎支原體FH株基因組DNA為模板,利用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增獲得黏附蛋白P1基因羧基末端部分編碼序列,再將其克隆到克隆載體pEASY-T1上,進(jìn)而得到了pEASY-MP-P1F這一重組質(zhì)粒。后續(xù)實(shí)驗(yàn)對(duì)pEASY-MP-P1F進(jìn)行基因測(cè)序檢查,把測(cè)序結(jié)果正確的P1F基因克隆到表達(dá)載體pET32a(+)上,構(gòu)建pET32a-MP-P1FE這一重組表達(dá)質(zhì)粒。接著對(duì)pET32a-MP-P1FE中的P1FE進(jìn)行測(cè)序,檢測(cè)正確后轉(zhuǎn)化大腸桿菌BL21(DE3),利用IPTG誘導(dǎo)表達(dá),以Ni~(2+)親和層析柱對(duì)pET32a-MP-P1FE表達(dá)的重組蛋白進(jìn)行純化,SDS-PAGE顯示純化的重組蛋白相對(duì)分子質(zhì)量為50kDa至60kDa,與預(yù)計(jì)值55.9kDa一致。Western Blotting、ELISA免疫檢測(cè)證實(shí),重組蛋白與感染肺炎支原體病人血清中的抗體有特異性反應(yīng)。因此,此次研究成功克隆表達(dá)了具有免疫原性的黏附蛋白P1羧基末端基因,為進(jìn)一步研究開發(fā)肺炎支原體診斷試劑和疫苗等奠定了良好的基礎(chǔ)。
[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.
【學(xué)位授予單位】：湖南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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