截短弓形虫表面抗原SAG2C的基因克隆、蛋白表达及其初步应用

Cloning and expression of a recombinant truncated form of the surface antigen SAG2C from Toxoplasma gondiiand its preliminary use

目的克隆截短的弓形虫表面抗原SAG2C基因,在大肠埃希菌中表达SAG2C蛋白,并探讨其在弓形虫病诊断中的应用。方法对已知的弓形虫SAG2C基因序列进行部分取舍,用RT-PCR技术从弓形虫Prugniaud(PRU)株的总RNA中扩增截短的SAG2基因片段,插入载体pET32a(+)中,转化大肠埃希菌BL21,IPTG诱导表达,应用Western blot和ELISA检测重组表达蛋白的免疫反应性。用重组SAG2C蛋白ELISA法检测弓形虫感染血清特异抗体,观察初步应用效果。结果从弓形虫PRU株总RNA中扩增出截短的SAG2C基因片段,成功构建了重组表达质粒pET32a(+)-tSAG2C;该重组质粒经IPTG诱导能表达可溶性大小为51ku的SAG2C蛋白。Western blot显示重组SAG2C能被弓形虫感染小鼠血清识别;以重组SAG2C蛋白、重组SAG1蛋白及BAG1蛋白ELISA检测精神病患者血清弓形虫抗体,阳性率分别为8.07%(23/285)、4.56%(13/285)和7.37%(21/285),差异无统计学意义(P>0.05)。结论成功构建了重组质粒pET32a(+)-tSAG2C,表达的融合蛋白具有免疫反应性,具有用于弓形虫感染诊断的潜在价值。

Objectives To clone a truncated form of the surface antigen 2C gene （SAG2C） of Toxoplasma gondii, ex press it in E. toll, and then investigate the use of the recombinant protein to diagnose toxoplasmosis. Methods A trun- cated form of the SAG2C sequence was amplified from the total RNA of a PRU strain of T.gondii using RT-PCR. The amplified fragment was then subcloned into the expression vector pET32a （＋） and expressed in E.coliafter induction with IPTG. The immunoreactivity of the recombinant protein was analyzed using Western blotting and ELISA. The re combinant protein was used in ELISA to identify antibodies in sera from patients with a mental illness, and the effective ness of their preliminary use was determined. Results The expression product was analyzed using Sf）S-PAGE. Results indicated that the truncated SAG2C gene was expressed in a soluble form in E. colt and that the protein had a molecular weight of 51 ku. Western blot analysis revealed that the recombinant tSAG2C was recognized by sera from mice with tox- oplasmosis. ELISA with the recombinant SAG2C, recombinant SAG1, and recombinant BAG1 was used to detect anti bodies in sera from patients with a mental illness. Sera tested positive at a rate of 8.07% （23/285）with recombinant SAG2C, a rate of 4. 56% （13/285）with recombinant SAG1, and a rate of 7. 37%0 （21/285） with recombinant BAG1. ELISA results for the three genes did not differ significantly（P）〉0.05）. Conclusion A truncated form of the SAG2C gene was cloned and expressed as a soluble fusion protein in E. colt. The recombinant protein displayed specific immuno reactivity, so il may be used as a diagnostic antigen to detect T. jzondii infection.