肺炎支原体P1黏附蛋白基因的克隆与表达

Cloning and expression of the gene encoding the P1 adhesion protein of Mycoplasma pneumoniae

目的对肺炎支原体(Mycoplasmapneumoniae,Mp)3’端的P1黏附蛋白基因片段进行基因克隆、重组表达、蛋白纯化和表达产物的免疫性分析,为临床诊断试剂和疫苗的研制打下基础。方法应用PCR技术直接从MpFH株染色体DNA中扩增894bp的3’端p1基因片段(p1’),将此基因片段插入表达载体pGEX-6P-1后,转入大肠杆菌BL21扩增后提取重组质粒进行酶切、PCR扩增及核苷酸测序进行鉴定。诱导阳性克隆株表达重组蛋白后纯化,用兔抗Mp多克隆血清通过免疫印迹实验鉴定其免疫反应性。结果插入重组质粒的p1基因片段经测序后,与GenBank中p1基因核苷酸序列(AF290001、AF290002、AF286371、AE000002、M21519、M18639)比较,其同源性为99.66%～100%。氨基酸序列同源性为99.32%～100%。经SDS-PAGE分析,重组蛋白的相对分子量约为59kDa。免疫印迹实验证实重组蛋白是P1黏附蛋白抗原。结论成功构建了pGEX-6P-1-p1’重组质粒并获得表达。此p1’基因重组质粒表达的蛋白具有免疫反应性,有希望成为特异性抗原诊断试剂和疫苗的候选抗原。

The 3' terminal end of the P1 adhesin gene fragment of Mycoplasma pneumoniae (Mp) was cloned, expressed and purified, and its immuno-reactivities of the expressed products were analyzed in the present investigation,in order to provide the basis for diagnosis and vaccine development. In this study, the 3'end of the P1 gene fragment (p1') of the chromosomal DNA in FH strain of Mp was amplified with PCR, and inserted into the vector pGEX-6P-1. The recombinant plasmid was used to transform competent E.coli strain BL21 by IPTG induction, and underwent the processes of enzyme digestion, PCR amplification, nucleotide sequencing and purification. Western blotting assay was used to determine the immuno-reactivity of the purified recombinant proteins. The experimental results showed that the homology of the p1'gene with the gene sequences reported in GenBank (AF290001, AF290002, AF286371, AE000002, ,M21519, M18639) was 99.66%～100%, and the homogeneity in the corresponding amino acids was 99.32%～100%. A specific recombinant protein with molecular weight of 59 kDa was found to be expressed as demonstrated by SDS-PAGE analysis, and this protein was proved to be P1 adhesin antigen, as analyzed by Western blotting. It concludes that the 3' end gene fragment with expected antigenic determinant of P1 adhesin of M.pneumoniae is expressed in E.coli BL21 cells, and the corresponding recombinant protein shows definite immuno-reactivity, suggesting that it might be used as a novel diagnostic reagent and suitable candidate antigen for vaccine development.