沙眼衣原体LpxA蛋白的表达、纯化及多克隆抗体的制备

The expression of LpxA of Chlamydia trachomatis and preparation of its polyclonal antibody

目的纯化沙眼衣原体LpxA蛋白并制备抗LpxA蛋白的多克隆抗体,为研究LpxA蛋白的功能奠定基础。方法 PCR扩增LpxA基因,以pET28a质粒为载体,构建表达质粒pET28a-LpxA,转化大肠埃希菌BL21,利用IPTG诱导含有6*His的融合蛋白表达,并使用Ni 2+亲和层析柱进行纯化。以纯化的His-LpxA蛋白作为免疫原,经背部皮下免疫新西兰兔,制备多克隆抗体,并使用免疫印迹法检测抗体与His-LpxA蛋白的反应以及使用ELISA法测定多克隆抗体的滴度。结果重组表达质粒pET28a-LpxA构建成功,融合His-LpxA蛋白的相对分子质量为32.8kDa,能够在大肠杆菌中高效表达,纯化后目的蛋白纯度约为95%,免疫新西兰兔后,抗血清能够识别重组的His-LpxA蛋白,其滴度大于1∶10 240。结论成功纯化了LpxA蛋白和制备了抗血清,为研究LpxA蛋白的功能提供实验基础。

Objective To express and purify recombinant LpxA of Chlamydia trachomatis in E.coli,and prepare antiserum against LpxA for immunoassays.Methods The gene LpxA was amplified by PCR,and subcloned into the pET28 ato generate a recombinant plasmid pET28 a-LpxA and express 6＊His-LpxA tagged fusion protein.Then the plasmid was transformed into E.coli BL21 which were induced by IPTG.The 6＊His-LpxA fusion protein was purified through Ni 2＋-chelating affinity chromatography.The purified 6＊His-LpxA fusion protein was used as an immunogen to inject into rabbit to produce antiserum.The immunoblotting was used to detect the reactions of antibodies and HisLpxA protein.The Elisa was used to determine the titer of polyclonal antibodies.Results The recombinant plasmid pET28 a-LpxA was constructed successfully.The 32.8 kDa fusion His-LpxA protein was highly expressed in E.coli.After purified,the purity of the target protein is approximately 95%.After immunization with New Zealand rabbits,the antiserum can recognize the recombinant protein.His-LpxA protein with a titer greater than 1∶10 240.ConclusionRecombinant LpxA protein was successfully purified which induced high antibody level in rabbits.