刚地弓形虫RH株主要表面抗原1的原核表达和纯化及免疫反应性检测

The evaluation expression purification and immunoresponse of the major surface antigen SAG1 of Toxoplasma gonddi RH strain

目的 对重组的刚地弓形虫RH株主要表面抗原 1(SAG1)原核表达载体SAG1/pET 32c ,在大肠杆菌中诱导表达产物进行亲和纯化 β免疫反应性检测评价。 方法 接种含SAG1/pET 32c的BL2 1单菌落至LB肉汤中 ,1∶10 0稀释转种后用 1mmol/L终浓度的IPTG诱导表达 ,表达产物用Ni2 + 螯合柱亲和纯化 ,并用免疫印迹技术对纯化的融合蛋白进行免疫反应性检测 ,用重组融合蛋白对弓形虫病 5 2份阳性和 4 0份阴性血清进行初步检测。结果 SAG1/pET 32c在原核系统中 ,经诱导表达出约 370 0 0大小的融合蛋白 ,并以包涵体的形式存在 ;通过Ni2 + 亲和柱纯化可获得SAG1重组融合蛋白。免疫印迹试验表明 ,重组SAG1融合蛋白能被弓形虫阳性血清特异性的识别。初步检测表明 ,阳性和阴性符合率分别为 81% (42 /5 2 )和 95 % (38/40 )。结论 SAG1在原核系统获得高效表达 ,亲和纯化的rSAG1具有较强的免疫反应性 ,对弓形虫病的免疫诊断具有潜在应用价值 ,也为人用或兽用的弓形虫病免疫诊断试剂盒研制奠定了基础。

Objective To express the major surface antigen gene SAG1 in E. coli, purify the recombinant fusion protein SAG1 and evaluate its immunoresponse. Methods A single colony of E. coli BL21 containing the plasmid SAG1/pET-32c was inoculated LB broth, then diluted 1∶100 into 1000 ml LB broth and induced with 1mmol/L IPTG. The recombinant SAG1 was purified with Ni 2+ chelating HiTrap HP column. Its immunoresponse was evaluated with Western-blot. Fifty-two IgG-positive and forty IgG-negtive human sera were tested with rSAG1.Results The recombinant SAG1 protein about 37 kDa was expressed in E.coli as inclusion body. rSAG1 was prepared with Ni 2+ column purification. The result of Western-blot showed that rSAG1 could be recognized by positive serum of Toxoplasmosis. The positive correspondence rate and negtive correspondence rate of rSAG1-ELISA was 81% (42/52) and 95% (38/40) respectively. Conclusions High-level expression of SAG1 was achieved in E. coli, the purified rSAG1 had strong immunoresponse. It indicated that rSAG1 had a potential value for immunodiagnosis of Toxoplasmosis.