鹦鹉热衣原体Mip基因原核表达载体的构建及多克隆抗体的制备  

Construction of prokaryotic vector encoding Chlamydia psittaci Mip gene and the preparation of its polyclonal antibodies

作　　者：胡春生[1] 符玺宗 周鹏[2] 柏琴琴[2] 曾心靛 周朴帆 刘子卿 张程[1] 陈丽丽[2] 

机构地区：[1]湖南省疾病预防控制中心,湖南长沙410005 [2]南华大学公共卫生学院

出　　处：《实用预防医学》2017年第3期280-283,共4页Practical Preventive Medicine

基　　金：国家自然科学基金项目(81572011);湖南省自然科学基金(2016JJ3103);湖南省研究生科研创新项目(CX2016B482)

摘　　要：目的克隆鹦鹉热衣原体巨噬细胞感染增强蛋白(Microphage infectivity potentiator,Mip)基因,构建原核重组质粒,诱导表达重组蛋白,并免疫BALB/c小鼠,制备多克隆抗体,为进一步研究其功能奠定了基础。方法提取鹦鹉热衣原体6BC基因组DNA,PCR扩增Mip基因,克隆至p ET-30a(+),构建原核表达载体p ET-30a(+)-Cps Mip。PCR、酶切及测序鉴定后,IPTG诱导p ET-30a(+)-Cps Mip在E.coli BL21中表达目的蛋白。融合蛋白纯化后免疫BALB/c小鼠,ELISA检测小鼠血清中抗Mip抗体的效价。结果 PCR扩增得到大小约为768 bp左右的Mip目的片段;经PCR、酶切及测序鉴定,证明构建的原核重组质粒中插入的片段为Cps Mip基因,测序结果经BLAST分析,与Genbank上登录序列完全一致。经IPTG诱导,重组工程菌表达一相对分子量(Mr)约为34 k D的目的蛋白;ELISA检测免疫小鼠血清效价为1:128 000。结论成功构建了鹦鹉热衣原体Mip基因原核表达载体,并表达了相应可溶性蛋白,制备了高效价的鼠抗Mip多克隆抗体。Objective To construct a prokaryotic recombinant plasmid containing the gene encoding microphage infectivity potentiator （Mip） of Chlamydia psittaci （C. psittaci）, and lay a basis for further study on the function of the recombinant protein. Methods The C. psittaci 6BC Mip gene was amplified by PCR and cloned into plasmid pET-30（＋）, then transformed into E.coli BL21. After being identified by PCR, restriction enzymes cleavage and nucleotide sequencing, the positive strains were inducted by IPTG. Then the recombinant protein Mip was analyzed by SDS-PAGE and purified with Ni-NTA-His affinity chromatography. The purified recombinant Mip was used to immunize BALB/c mice, and the antibodies to Mip in mice sera were titrated with ELISA. Results A 768bp fragment of C. psittaci Mip gene was obtained by PCR. The target gene was proved to be successfully inserted into pET30（＋） by PCR, restriction enzymes cleavage and nucleotide sequencing. The similarity was 100% between the inserted gene and Mip gene reported in Genbank by BLAST analysis. After the induction and purification, a recombinant protein about 34KD was obtained. Specific humoral response was elicited in BALB/c mouse after immunization with the purified recombinant protein and the antibody titer in mice’ sera was higher than 1：128,000. Conclusions Prokaryotic expression plasmid pET-30（＋）-Cps Mip is successfully constructed, a molecular weight about 34 KDa protein is obtained and specific antibody is successfully prepared.

关 键 词：鹦鹉热衣原体 MIP基因 免疫原性 多克隆抗体 

分 类 号：R374.2[医药卫生—病原生物学]