弓形虫表面抗原SAG2基因片段的克隆与原核表达

目的　扩增弓形虫主要表面抗原 (SAG2 )编码基因片段并进行重组表达。方法　设计合成 1对引物 ,从弓形虫基因组DNA中扩增SAG2基因序列 ,以低熔点琼脂糖回收纯化 ,并以限制性内切酶BamHⅠ和SalⅠ进行双酶切、纯化后 ,再插入表达载体 pGEX - 4T - 2 ,经PCR和双酶切筛选 ,测序验证后 ,在大肠杆菌中进行表达 ,并用SDS -PAGE和Westernblot鉴定。结果　从弓形虫核酸提取物中扩增出约 477bp的SAG2基因 ,构建成功了重组质粒 pGEX - 4T - 2 -SAG2 ;SAG2基因在大肠杆菌中得到高效表达。SDS -PAGE电泳 pGEX - 4T - 2 -SAG2的融合蛋白条带的分子量约为 42kD ,Westen -blot显示融合蛋白能被兔抗弓形虫血清识别。结论　GST融合表达载体的构建和SAG2基因片段成功表达 ,为进一步为SAG2重组疫苗及重组诊断抗原的研制奠定了基础。

Cloning and prokaryotic expression of SAG2 gene of Toxoplasma gondii

Aim To amplify SAG2 gene of Toxoplasma gondii and express SAG2 fusioned with GST Methods The SAG2 gene fragment was amplified by PCR After purification and digestion with BamHⅠ and SalⅠ,the gene fragment was ligated to an expression vector pGEX 4T 2 Screening positive recombinants sequenced were induced for expression,which was subsequently detected by SDS PAGE and Western blot Results and Conclusion SAG2 gene was amplified,and the recombinant plasmid pGEX 4T 2 SAG2 and was constructed successfully The results of SDS PAGE and Western blot revealed that the molecular weight of recombinant protein pGEX 4T 2 SAG2 was 42KD and GST fusion protein was confirmed by the positive serum of rabbit infected by RH strain The construction of pGEX 4T 2 SAG2,together with the recombinant protein can lay a base for further application to diagnosis and vaccination