沙眼衣原体类噬菌体衣壳蛋白CtF的表达及多克隆抗体制备  

Expression and polyclonal antibodies preparation of the phage capsid protein analogue CtF encoded in Chlamydia trachomatis

作　　者：王秋平[1] 李琪 欧瑶琪 朱冰玉 周鹏[2] 陈丽丽[2] 柏琴琴[2] Qiuping Wang;Qi Li;Yaoqi Ou;Bingyu Zhu;Peng Zhou;Lili Chen;Qinqin Bai(The First Affiliated Hospital of University of South China Clinical Laboratory,Hengyang 421001,China;College of Public Health,University of South China,Hengyang 421001,China)

机构地区：[1]南华大学附属第一医院检验科,湖南衡阳421001 [2]南华大学公共卫生学院,湖南衡阳421001

出　　处：《生物技术》2018年第4期377-381,共5页Biotechnology

基　　金：湖南省教育厅科学研究项目("多重耐药鲍曼不动杆菌噬菌体的分离鉴定及其对生物被膜的裂解作用";No.16C1390);南华大学大学生研究性学习和创新性实验计划("微小噬菌体phiCt5的衣壳蛋白VP1对沙眼衣原体生长的影响";No.2017XJYZ056)

摘　　要：[目的]预测沙眼衣原中类噬菌体衣壳蛋白CtF的空间结构,表达重组蛋白并制备多克隆抗体。[方法]采用SOPMA和I-TASSER等软件预测CtF的空间结构,利用MEGA6分析其系统发育关系;构建重组质粒pET-28a-CtF,转化至E.coli BL21(DE3)中进行克隆表达;纯化重组蛋白免疫ICR小鼠制备多克隆抗体。[结果]CtF蛋白包含八股β桶状核心结构和环形延伸结构,与已知的衣原体噬菌体VP1蛋白遗传距离较近;重组质粒表达相对分子量约为64 kDa的融合蛋白;免疫小鼠获得免疫血清效价达1∶12 800。[结论]CtF蛋白疑似为沙眼衣原体噬菌体的衣壳蛋白;重组质粒表达相对分子量约为64 kDa蛋白,该蛋白免疫原性良好,为进一步研究其功能奠定了基础。[Objective] To predict structure of phage capsid protein analogue CtF,express a recombinant form of the protein r CtF,and prepare its polyclonal antibodies. [Methods] Online software such as SOPMA and I-TASSER were used to predict its secondary and tertiary structures. Phylogenetic tree of CtF protein was constructed through MEGA6 software. The prokaryotic expression vector pET-28 a-CtF was constructed and transformed into the E. coli BL21（ DE3） competent cell to express a recombinant form of the protein r CtF. Then the purified recombinant r CtF protein was used to immunize ICR mice to prepare its polyclonal antibodies. [Results] The CtF protein harbors an eight-stranded beta-barrel core structure and a loop extension similar to other Gokushovirinae members. Phylogenetic analysis of CtF protein revealed a closer genetic distance with the VP1 capsid proteins of chlamydiaphages. A molecular mass of approximately 64 k Da protein was efficiently expressed by the prokaryotic expression vector pET-28 a-CtF. And the antibody titer in sera of mice was as high as 1∶ 12 800. [Conclusion]The CtF protein may be the capsid protein of Chlamydia trachomatis phages. The recombinant form of protein r CtF about 64 kDa was successfully obtained and has good immunogenicity,which will provide a basis for further study on the function of the recombinant protein.

关 键 词：沙眼衣原体 类噬菌体衣壳蛋白 生物信息分析 表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学]