鹦鹉热衣原体噬菌体Chp1衣壳蛋白VP1的原核表达及多克隆抗体制备  

Prokaryotic expression of the capsid protein VP1 of Chlamydiapsittaci bacteriophage Chp1 and preparation of its polyclonal antibodies

作　　者：李琪 王秋平[2] 周鹏[1] 陈丽丽[1] 柏琴琴[1] LI Qi;WANG Qiu-ping;ZHOU Peng;CHEN Li-li;BAI Qin-qin(College of Public Health,University of South China,Hengyang 421001,Hunan,China;Clinical Laboratory,The First Hospital Affiliated with the University of South China)

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

出　　处：《中国病原生物学杂志》2018年第9期975-978,983,共5页Journal of Pathogen Biology

基　　金：湖南省自然科学基金项目(No.2016JJ3103);湖南省教育厅科学研究项目(No.16C1390)

摘　　要：目的原核表达并纯化鹦鹉热衣原体(Chlamydia psittaci,Cps)噬菌体Chp1衣壳蛋白VP1的重组蛋白rVP11-190,并制备多克隆抗体。方法采用Clustal Omega在线软件对6株衣原体噬菌体VP1蛋白的氨基酸序列进行多重比对,确定Chp1VP1蛋白的保守区和特异区,并用DNAStar软件预测抗原表位。选择VP11-190与pET28a(+)载体连接构建原核表达载体pET28a-VP11-190,转化至E.coli BL21(DE3)中诱导表达,采用Ni-NTA亲和层析法纯化重组蛋白;用纯化的重组蛋白免疫ICR小鼠,制备免疫血清,ELISA法检测其抗rVP11-190的效价。结果构建的原核表达载体pET28(a)-VP11-190经IPTG诱导高效表达了相对分子质量约为30×103的重组蛋白。用该蛋白免疫小鼠,制备多克隆抗体血清,ELISA法测定免疫小鼠血清抗体效价为1∶12 800。结论成功原核表达并纯化了噬菌体Chp1衣壳蛋白VP11-190的重组蛋白,制备的鼠抗VP11-190多克隆抗体血清效价及特异性良好,为进一步分离鉴定鹦鹉热衣原体的噬菌体奠定了基础。Objectives To express the recombinant protein rVP11-190 of Chlamydia psittaci phage Chp1 capsid protein VP1 in a prokaryotic expression system,to purify that protein,and to prepare polyclonal antibodies against it. Methods The online software Clustal Omega was used to align the amino acid sequences of capsid protein VP1 from 6 chlamydiaphages and to determine the conserved and specific regions of Chp1 capsid protein VP1.The software DNAStar was used to predict its antigenic epitopes.VP11-190 was ligated into pET-28 a（＋）to construct the prokaryotic expression plasmid pET28 a-VP11-190.The recombinant plasmid was then transformed into E.coli BL21（DE3）.Expression of the protein was induced,and the recombinant protein was purified using Ni-NTA affinity chromatography.The recombinant protein was used to immunize ICR mice to prepare immune serum.The titer of antibodies to rVP11-190 in mice sera was detected using ELISA. Results The constructed prokaryotic expression vector pET28（a）-VP11-190 highly expressed a recombinant protein with a molecular mass of about 30 ×10^3 in response to IPTG.Mice were immunized with this recombinant protein to prepare polyclonal antibody serum,and the antibody titer of immunized mice was 1：12 800 according to ELISA. Conclusion The recombinant protein of the capsid protein VP11-190 of phage Chp1 was successfully expressed and purified,and the prepared mouse anti-VP11-190 polyclonal antibody had a good serum titer and specificity.This work has laid the foundation for further isolation and identification of C.psittaci phages.

关 键 词：鹦鹉热衣原体 噬菌体Chp1 VP1蛋白 原核表达 多克隆抗体 

分 类 号：R374[医药卫生—病原生物学]