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

[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.