鼠肺炎沙眼衣原体质粒蛋白pgp5的克隆表达鉴定及免疫原性分析

Cloning Expression and Evaluation of Immunogenicity of Plasmid-encoded pgp5 Protein of Chlamydia Muridarum

目的获取鼠肺炎沙眼衣原体(Chlamydia muridarum)质粒蛋白pgp5的基因及纯化的蛋白并鉴定其免疫原性。方法设计引物,PCR扩增目的基因,将其定向插入到原核表达载体pET28a中,然后将重组质粒转化入大肠杆菌E.coli DH5α中,并用PCR扩增及序列测定等方法对重组质粒进行鉴定。再将重组质粒转化入感受肽Rosetta(DE3)并诱导表达,用镍柱纯化pgp5-his融合蛋白。用纯化后的目的蛋白免疫新西兰家兔,酶联免疫吸附试验(ELISA)测定抗体效价,Western blot、细胞免疫荧光方法检测抗体与pgp5蛋白的结合。结果所获得的pgp5基因片段经测序长度为795 bp,检索确认其序列与GeneBank一致。十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹实验均显示获得相对分子质量约29 000的纯化蛋白。ELISA检测多克隆抗体效价达1∶100 000。细胞免疫荧光检测结果显示抗体可与体外培养的鼠肺炎沙眼衣原体特异性结合。结论成功表达pgp5-his融合蛋白,并制备了高效价、高特异性的抗pgp5抗体,为进一步研究奠定了基础。

Objective To clone the plasmid-encoded pgp5 of Chlamydia muridarum(CM),and evaluate its immunogenicity.Methods Primers were designed,and PCR was used to amplify the gene of plamsid-encoded pgp5.The gene was inserted into the prokaryotic expression vector pET28 a,and then the recombinant plasmid was transformed into E.coli DH5α,followed by sequencing and PCR identification.After the identification,the recombinant plasmid was transformed into Rosetta(DE3)to induce expression of pgp5.ANi-ion affinity chromatography column was used to purify the pgp5-his fusion protein,which was used to immunize New Zealand rabbits for preparation of polyclonal antibodies.The antibody titer was determined by enzyme-linked immunosorbent assay(ELISA),and the binding of the antibody to pgp5 protein was detected by Western blotting and immunofluorescence.Results The length of pgp5 gene was 795 bp,consistent with pgp5 of Chlamydia muridarum in GeneBank.The molecular weight of the purified target proteins was 29 000 determined by SDS-PAGE and Western blotting.Meanwhile,the titer of anti-pgp5 polyclonal antibodies was 1∶100 000 detected by ELISA.Conclusion The pgp5-his fusion protein has been successfully expressed,and a high-titer,highly specific anti-pgp5 antibody is prepared,which lay a foundation for further research.