豚鼠结膜炎衣原体噬菌体衣壳蛋白Vp3的克隆、表达和鉴定  

Cloning, expression and identification of phage＇s capsid Vp3 protein of guinea pig inclusion conjunctivitis chlamydia

作　　者：刘原君[1] 姚卫锋[1] 侯淑萍[1] 齐蔓莉[1] 王惠平[1] 刘全忠[1] 

机构地区：[1]天津医科大学总医院皮肤性病科天津性传播疾病研究所,300052

出　　处：《中华传染病杂志》2010年第12期712-715,共4页Chinese Journal of Infectious Diseases

基　　金：基金项目：国家自然科学基金资助项目（30671879）

摘　　要：目的获得豚鼠结膜炎衣原体（GPIC）噬菌体~CPGl衣壳蛋白Vp3基因及其蛋白。方法提取φCPG1及其核酸，PCR扩增Vp3基因，双酶切后将其定位插入原核表达载体pET30a（＋）中，构建重组表达质粒，转化人大肠埃希菌BL-21，并酶切分析、PCR扩增及测序鉴定，十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及Western印迹鉴定，胶回收纯化。结果获得的Vp3基因片段经测序，长度为447bp，检索确认其序列与GenBank一致。对重组质粒进行诱导表达，SDS-PAGE和Western印迹均显示获得相对分子质量约25000的GPIC噬菌体φCPC1衣壳蛋白Vp3，并得到了纯化蛋白。结论成功表达了噬菌体φCPG1衣壳蛋白Vp3，为进一步研究其作用机制和临床应用奠定基础。Objective To get phagers capsid Vp3 gene and protein of guinea pig inclusion conjunctivitis （GPIC） chlamydia. Methods The genome DNA was extracted from the φCPG1 phage. The full sequence of Vp3 gene was amplified by polymerase chain reaction （PCR） from the above genome DNA. The Vp3 gene was digested by restriction endonuclease and then inserted into prokaryotic plasmid vector pET30a （＋）. The recombinant plasmid was transformed into E. coil BL21, and was identified by restriction endonuclease, PCR and sequencing. The E. coil BL21 with expected recombinant plasmid was induced and the expressed recombinant Vp3 protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, then purified by agarose gel. Results The recombinant gene was sequenced and proved to be 447 bp which was consistent with the φCPG1 Vp3 gene sequence in GenBank. A 25 000 capsid protein was expressed and confirmed by SDS PAGE and Western blot. The purified protein was obtained. Conclusion The capsid Vp3 protein of φCPG1 is successfully expressed and purified, which is helpful for the further study on its mechanism and clinical applications.

关 键 词：衣原体 鼠 细菌噬菌体 衣壳蛋白质类 重组 遗传 基因表达 质粒 

分 类 号：R686[医药卫生—骨科学]