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目的重组表达肺炎衣原体Cpn0147基因,并对表达的内源性蛋白进行细胞内定位,对重组蛋白进行生物信息学分析。方法使用PCR方法从肺炎衣原体菌株AR39基因组中扩增第0147段开放读码区基因,使用限制性内切酶BamHⅠ和NotⅠ酶切目的片段和载体PGEX-6P2,T4连接酶连接后转化入大肠埃希菌感受态XL-Blue,并诱导表达融合蛋白GST-Cpn0147。用融合蛋白免疫小鼠,制备抗体,应用IFA方法对衣原体感染细胞内Cpn0147基因表达的内源性蛋白进行定位。利用软件Expasy对重组蛋白进行生物信息学分析。结果克隆出肺炎衣原体基因Cpn0147,全长450 bp,编码蛋白分子质量单位为14.727 ku,表达的融合蛋白GST-Cpn0147分子质量单位约为43 ku;用制备的抗体做IFA,显示现该蛋白定位于肺炎衣原体包涵体膜上。生物信息学分析该蛋白含2个抗原决定簇,具有良好的抗原性和亲水性。结论肺炎衣原体Cpn0147基因编码蛋白为一包涵体膜蛋白,抗原性和亲水性较好。
Objective To recombinantly express Cpn0147 gene of Chlamydia pneumoniae and to locate the expressed endogenous protein in vivo. The recombinant protein was analyzed by bioinformatics. Methods The open reading frame (ORF) of the first 0147 was amplified from the genome of Chlamydia pneumoniae strain AR39 by PCR. The target fragment was digested with restriction endonucleases BamH Ⅰ and Not Ⅰ and the vector pGEX-6P2 was ligated and ligated into E. coli Xylobacterium competent XL-Blue, and induced the expression of the fusion protein GST-Cpn0147. Mice were immunized with the fusion protein to prepare antibodies, and the endogenous protein expression of Cpn0147 gene in Chlamydia trachomatis-infected cells was localized by IFA method. The use of software Expasy bioinformatics analysis of recombinant proteins. Results The Cpn0147 gene of Chlamydia pneumoniae was cloned and its full length was 450 bp. The molecular weight of the expressed protein was 14.727 ku. The molecular mass unit of the expressed protein GST-Cpn0147 was about 43 ku. IFA was performed on the prepared antibody, Chlamydia pneumoniae inclusion body membrane. Bioinformatics analysis of the protein contains two epitopes, has good antigenicity and hydrophilicity. Conclusion The Cpn0147 gene of Chlamydia pneumoniae is a inclusion body membrane protein with good antigenicity and hydrophilicity.