弓形虫磷酸丙糖异构酶原核表达及免疫保护的初步研究

目的克隆刚地弓形虫磷酸丙糖异构酶(Triosephosphate isomerase,TPI)基因,原核表达和纯化蛋白,研究其对弓形虫感染小鼠的免疫保护作用。方法抽提弓形虫速殖子总RNA,利用PCR技术扩增刚地弓形虫TPI基因,构建重组原核表达载体TPI/p ET-28a(+),转化大肠杆菌BL21,IPTG诱导目的蛋白表达,镍亲和层析法纯化目的蛋白。将与佐剂乳化后的TPI蛋白对小鼠颈背部多点皮内免疫,间隔2周,连续免疫4次,最后一次为加强免疫,同时设置佐剂对照组与正常对照组。尾静脉采血检测小鼠血清抗体效价。以400个弓形虫速殖子/只接种小鼠腹腔,观察记录各组小鼠的生存率。结果成功扩增到弓形虫TPI基因,构建原核表达载体TPI/p ET-28a(+),获得纯化的TPI蛋白,TPI免疫4次后的小鼠血清抗体效价可达1:100 000以上,且生存期显著高于佐剂对照组与正常对照组。结论弓形虫TPI蛋白对弓形虫感染小鼠具有一定的免疫保护作用,为研究弓形虫疫苗奠定了理论基础。

Preliminary research on prokaryotic expression and immune protection of triosephosphate isomerase of Toxoplasma gondii

Objective To study the prokaryotic expression and immune protection of triosephosphate isomerase （TPI） of Toxoplasma gondii in mice. Methods Total RNA was extracted from toxoplasma tachyzoites， and TPI fragment was amplified by PCR and cloned into the prokaryotic expression vector pET?28a （+）. The target protein was induced with IPTG and purified by Ni?NTA affinity chromatography. The mice were immunized 4 times by emulsified TPI with adjuvant， and the last time was the strengthen immunization. At the same time， an adjuvant group and a normal group were set as controls. The blood samples were got from the tail vein of the mice， and the serum antibody titres were detected. All the mice were challenged with 400 toxoplasma tachyzoites to observe the survival time. Results The TPI gene was amplified from T. gondii cDNA by PCR. The recombinant vector TPI/pET?28a （+） was usefully constructed， and the TPI protein was expressed and purified. The serum antibody titre could be more than 100 thousand. After infected with toxoplasma tachyzoites， the survival time of the mice in the experimental group was longer than that of the mice in the control groups. Conclusion The TPI protein of T. gondii could trigger the immunoprotection against T. gondii challenge in the mice.