刚地弓形虫RH株主要表面抗原1的基因克隆及原核表达

Gene cloning and prokaryotic expression of the major surface antigen gene 1 of Toxoplasma gonddi RH strain

目的 从刚地弓形虫RH株基因组DNA中扩增出其主要表面抗原 1(SAG1)全长的基因编码序列 ,构建重组原核表达载体并在大肠埃希菌中进行表达 ,为进一步研制弓形虫病免疫诊断试剂盒打下基础。方法 根据弓形虫RH株主要表面抗原SAG1的cDNA序列设计一对引物 ,利用聚合酶链反应 (PCR )技术从RH株弓形虫基因组中扩增出SAG1的全长基因 ,将其克隆到载体pGEX 4T 3中 ,并通过基因测序加以证实 ;重新设计引物将其亚克隆至原核表达载体pET 3 2c中 ,在大肠埃希菌中经IPTG诱导表达。结果 从刚地弓形虫RH株基因组中成功扩增到 10 3 0bp的全长SAG1基因 ,该基因在原核系统中经诱导表达出一分子量约 3 70 0 0大小的融合蛋白 ,表达产物以包涵体的形式存在。结论 SAG1是弓形虫主要的表面抗原 。

Objective To amplify the major surface antigen gene SAG 1 from genome DNA of Toxoplasma gonddi RH strain, construct recombinant procaryotic expression vector and express it in E.coli. Methods A pair of primers were designed according to cDNA sequence of SAG1, then the SAG1 gene amplified by PCR was cloned into the vector pGEX-4T-3 and proved by DNA sequencing. The SAG1 gene was subcloned into prokaryotic expression vector pET-32c, its expression was induced by IPTG. Results The SAG1 gene was successfully amplified from genome DNA of Toxoplasma gonddi RH strain and a 37 000 fusion protein was expressed in E.coli expression system pET-32c as inclusion body. Conclusions SAG1 was a major antigen in the surface of Toxoplasma, the SAG1 fusion protein has a potential value in diagnosis of Toxoplasmosis.