刚地弓形虫ME49株AP核酸内切酶的原核表达及其多克隆抗体的制备

Prokaryotic expression and purification of AP endonuclease from the ME49 strain of Toxoplasma gondii and preparation of polyclonal antibodies

目的原核表达、纯化刚地弓形虫(Toxoplasma gondii)ME49株AP核酸内切酶,并制备多克隆抗体。方法采用PCR方法扩增出刚地弓形虫ME49株DNA TGME49-216600(681bp)基因。将该片段分别插入到pET-28a和pGEX-4T-1中,构建重组表达质粒pET-28a-TGME49-216600和pGEX-4T-1-TGME49-216600并测序,转入大肠埃希菌BL21-CodonPlus后用IPTG诱导表达重组蛋白质。用His标签重组蛋白质免疫家兔,制备多克隆抗体,采用间接ELISA方法检测抗体效价;采用Western blot方法分析抗体特异性。结果 SDS-PAGE分析表达的His标签重组蛋白质分子质量单位约为35ku,GST标签重组蛋白质分子质量单位约为55ku。间接ELISA方法检测制备的兔抗His标签重组蛋白抗体效价>1∶16 000,Western blot方法验证显示该抗体能识别天然AP核酸内切酶。结论成功构建了TGME49-216600的表达载体,制备的多克隆抗体具有特异性,为刚地弓形虫ME49株AP核酸内切酶功能的研究奠定了基础。

Objectives To prokaryotically express and purify AP endonuclease from the ME49 strain of To.roplasma gondii and to prepare polyclonal antibodies. Methods The TGME49-216600 gene from the ME49 strain of T. gondii was amplified using PCR, resulting in a fragment about 681 bp in length. The fragment was then inserted into pET 28a and pGEX-4T-1 expression vectors to construct the recombinant plasmids pET-28a TGME49-216600 and pGEX-4T-1- TGME49-216600. Sequencing indicated that the plasmids were correct, and then the recombinant plasmids were cloned into BL21-CodonPlus. Expression of these recombinant proteins was induced with 0.01mmol/L of IPTG, and the proteins were purified with His GraviTrapTM and Glutathione-Sepharose 4B. Polyclonal antibodies against TGME49-216600 were obtained in rabbits immunized with the purified His-tagged recombinant proteins. Each rabbit was immunized every two weeks. The antibody titer was detected using an indirect ELISA assay and expressed as the optical density at A40-. The antibody specificity was detected with Western blotting. Results A TGME49-216600 fragment （681 bp in length） was amplified using PCR. Results of SD-PAGE indicated that the His-tagged TGME49-216600 recombinant protein had a molecular mass of about 35 ku and the GST-tagged TGME49 216600 recombinant protein had a molecular mass of about 55 ku. The TGME49 216600 antibody titer was higher than 1：16,000 according to an indirect ELISA assay. The natural protein was specifically recognized by polyclonal antibodies. Conclusion The TGME49-216600 gene was cloned and successfully expressed, and prepared polyclonal antibodies were specific to the protein. These findings could be used in follow up studies of the function of AP endonuelease in the ME49 strain of T. gondii.