鼠肺炎沙眼衣原体毒力因子Pgp3蛋白的分段克隆表达  

Cloning and expression of the different fragments of Pgp3 of Chlamydia muridarum

作　　者：徐宏俊 孙毅娜[2] 刘全忠[3] 刘原君[3] XU Hong-jun;SUN Yi-na;LIU Quan-zhong(Department of Dermatovenereology,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;Chu Hsien-I Memorial Hospital&Tianjin Institute of Endocrinology,Tianjin Medical University,Tianjin 300134,China;Department of Dermatovenereology,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]首都医科大学附属北京友谊医院皮肤性病科,北京100050 [2]天津医科大学朱宪彝纪念医院天津市内分泌研究所,天津300134 [3]天津医科大学总医院皮肤性病科,天津300052

出　　处：《临床和实验医学杂志》2021年第8期785-788,共4页Journal of Clinical and Experimental Medicine

基　　金：国家自然科学基金资助项目(编号:31570178);天津市自然科学基金资助项目(编号:16JCQNJC11100)。

摘　　要：目的获得鼠肺炎沙眼衣原体(CM)质粒编码蛋白Pgp3氨基端(n)、羧基端(c)和中间段(m)分别删除的蛋白Pgp3Δn、Pgp3Δm和Pgp3Δc,为近一步研究其功能及沙眼衣原体(C.t)致病机制提供基础。方法用聚合酶链式反应(PCR)扩增目的基因Pgp3Δn、Pgp3Δm和Pgp3Δc,分别将其定向插入原核表达载体PET-30a(+)中,连接产物转化入大肠埃希菌DH5α、涂板、挑取单克隆提取质粒进行酶切、测序鉴定。鉴定正确的重组质粒分别转化入大肠埃希菌BL21,15℃诱导表达16 h和37℃诱导表达4 h,考马斯亮蓝染色和蛋白质印迹法鉴定目的蛋白。结果PCR扩增的目的基因Pgp3Δn、Pgp3Δm和Pgp3Δc的长度分别为597、525、324 bp,NdeI和HindⅢ双酶切重组质粒后电泳显示酶切片段大小符合预期,测序结果显示插入片段序列均与基因库中一致;考马斯亮蓝染色和蛋白质印迹显示目的蛋白分子量分别约为22000、18700和12000,3个蛋白片段均在15℃诱导表达16 h的条件下表达量更多。结论成功表达Pgp3Δn-His、Pgp3Δm-His和Pgp3Δc-His融合蛋白,为进一步研究Pgp3的功能位点和作用机制奠定基础。Objective To get the different fragments of plasmid-encoded protein Pgp3 of Chlamydia muridarum:Pgp3Δn(deletion of the Pgp3 N-terminus),Pgp3Δm(deletion of middle domain of Pgp3)and Pgp3Δc(deletion of the Pgp3 C-terminus).Methods Polymerase chain reaction(PCR)was used to amplify the genes of Pgp3Δn,Pgp3Δm and Pgp3Δc,then these amplified genes were inserted into the prokaryotic expression vector of PET30 a(+),respectively.The products were transformed into DH5αcompetent cells,coated plates and were identified by enzyme digestion,sequencing.After the identification,the corrected recombinant plasmids were separately transformed into E.coli BL21 and the bacteria was induced for 16 h at 15℃or 4 h at 37℃.The expected proteins were identified by Coomassie brilliant blue staining and Western blotting.Results The length of amplified genes of Pgp3Δn,Pgp3Δm and Pgp3Δc were 597 bp,525 bp and 324 bp,respectively.Sequencing results show that the inserted fragment sequences are consistent with the sequences of Pgp3 in Genebank;Coomassiebright blue staining and Western blot showed that the molecular weight of the targetedproteins were about 22000,18700 and 12000,respectively.The better expression condition was at 15℃for 16 h.Conclusion The fusion proteins Pgp3Δn-His、Pgp3Δm-His and Pgp3Δc-His were successfully expressed and purified,which lays the foundation for further study.

关 键 词：鼠肺炎沙眼衣原体 Pgp3 基因表达 结构域 

分 类 号：R759[医药卫生—皮肤病学与性病学]