流产衣原体CPAF基因的克隆序列分析及原核表达  

Cloning,Sequence Analysis and Prokaryotic Expression of CPAF Gene of Chlamydia abortus

作　　者：刘萍 马晓霞 周小凯[1,2,3] 马鹏 常秋燕[1,2,3] 李林杰 李凌浩[1,2,3] 马忠仁 LIU Ping;MA Xiaoxia;ZHOU Xiaokai;MA Peng;CHANG Qiuyan;LI Linjie;LI Linghao;MA Zhongren(Key Laboratory of Bioengineering and Biotechnology of State Ethnic Affairs Commission,Northwest Minzu University,Lanzhou 730030,China;Gansu Engineering ＆ Technology Research Center for Animal Cell,Lanzhou 730030,China;Life Science and Engineering College,Northwest Minzu University,Lanzhou 730030,China)

机构地区：[1]西北民族大学生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]甘肃省动物细胞工程技术研究中心,甘肃兰州730030 [3]西北民族大学生命科学与工程学院,甘肃兰州730030

出　　处：《华北农学报》2018年第3期86-91,共6页Acta Agriculturae Boreali-Sinica

基　　金：中央高校基本科研业务费(31920170158);甘肃省科技计划项目(1504WKCA094);科技部援助项目(KY201501005)

摘　　要：为了研究流产衣原体蛋白酶样活性因子(CPAF)的免疫原性,建立快速有效的流产衣原体抗体检测方法,根据Gen Bank中公布的流产衣原体基因组序列,设计并合成了流产衣原体CPAF蛋白编码基因的特异性引物,以流产衣原体标准菌株基因组为模板,经PCR扩增获得长度为1 809 bp目的基因片段;对获得的目的基因进行测序,并用生物信息学软件进行预测分析;再将该片段插入原核表达载体p ET-30a中,经双酶切、测序鉴定;构建成功的表达质粒转化大肠杆菌BL21(DE3),并用IPTG对目的蛋白进行诱导表达,通过对诱导条件的优化,确定诱导表达的最佳条件,表达产物用SDS-PAGE和Western Blot检测。结果显示,成功克隆流产衣原体CPAF蛋白的编码基因,该编码产物可能是定位于宿主细胞质中的一种衣原体分泌性蛋白酶,分子内具有多个可能的抗原表位;重组表达质粒经IPTG诱导后,表达分子质量为67.6 ku的CPAF蛋白,该重组蛋白能与抗His-tag单克隆抗体以及流产衣原体阳性血清特异性结合,具有良好的反应原性。结果表明,以大肠杆菌表达系统重组表达的流产衣原体CPAF蛋白具有良好的免疫原性,可作为动物流产衣原体病的血清学诊断及亚单位疫苗研究的候选抗原。In order to investigate the immunogenicity of Chlamydial protease like activity factor（CPAF）of C. abortus and establish a rapid method for detection of Chlamydia abortus （ C. abortus ）,a pair of specific primers for CPAF of C. abortus were designed and synthesized. The gene was amplified by PCR from C. abortus reference strain. The obtained gene sequence was sequenced and analyzed by bioinformatics software. The obtained gene of 1 809 bp was inserted into plasmid pET-30a. The recombinant plasmid was identified by digesting and sequencing. Then the successfully constructed recombinant plasmid was transformed into E.coli BL21（DE3）strain as host cell for expression of the recombinant protein by IPTG induction. The production was detected by SDS-PAGE and Western Blot analysis. The CPAF gene of Chlamydia abortus was successfully cloned and analyzed. This gene was likely to be located in the cytoplasm of host cell,playing a role as a proteolytic enzyme with several potential antigenic epitope. As shown by SDS-PAGE and Western Blot analysis,a 67.6 ku recombinant protein was expressed by IPTG induction,which could combine with anti-His-tag monoclonal antibody and C. abortus positive serum with well reactiongenicity. These results suggested that the recombinant CPAF could be used as a potential antigen for serological tests and vaccine development of C. abortus .

关 键 词：流产衣原体 CPAF 克隆 序列分析 原核表达 

分 类 号：S855[农业科学—临床兽医学] Q78[农业科学—兽医学]