弓形虫复合基因SAG1-BAG1的构建、表达及重组抗原的免疫反应性分析

Construction,expression and identification of SAG1-BAG1 complex gene from Toxoplasma gondii

目的构建并表达弓形虫复合基因SAG1-BAG1及分析重组抗原的免疫反应性。方法利用PCR扩增弓形虫SAG1基因,利用RT-PCR扩增弓形虫BAG1基因,通过酶切连接分别将SAG1、BAG1基因克隆到pET28a表达载体中构建复合基因SAG1-BAG1。将质粒pET28a-SAG1-BAG1转入大肠杆菌BL21(DE3)中进行诱导表达,Western-blot分析该重组蛋白的免疫反应性。结果复合基因SAG1-BAG1全长约1 407 bp,重组蛋白相对分子质量约为50 000,表达量约占菌体总蛋白的10%,Western-blot分析显示重组蛋白具有良好的免疫反应性。结论成功构建并表达弓形虫复合基因SAG1-BAG1。

Objective To construct,express and identify SAG1-BAG1 complex gene from Toxoplasma gondii.Methods The SAG1 gene and BAG1 gene were amplified by PCR and RT-PCR,respectively.And the two genes were cloned into the same pET28a vector to construct SAG1-BAG1 complex gene.After induced by IPTG,the recombinant protein was identified by Western-blot.Results The SAG1-BAG1 complex gene was about 1 407 bp.The recombinant protein was expressed successfully,and was about 50 kDa in size.Western-blot showed that the protein could be identified specifically by Toxoplasma gondii infected serum.Conclusion The complex gene SAG1-BAG1 was constructed successfully.