弓形虫表面抗原SAG2重组蛋白的表达及纯化

目的在大肠埃希氏菌（E．coli）中表达弓形虫表面抗原2（SAG2），纯化制备重组蛋白rSAG2。方法采用聚合酶链反应（PCR）技术从弓形虫基因组中扩增出SAG2编码基因片段，以pMD-18T质粒作TA克隆，序列测定后亚克隆入表达载体pGEX-4T-2，并转化E．coliJMl09感受态菌，IPTG诱导表达rSAG2蛋白，重组rSAG2蛋白采用B—PER谷胱甘肽巯基转移酶（GsT）融合蛋白纯化试剂盒纯化并进行SDS—PAGE与免疫印迹（Western—blot）鉴定。结果SAG2编码基因扩增片段大小为469bp；测序结果显示，克隆的SAG2基因序列与GenBank中弓形虫RH株的同源序列（序列号GI：161925）完全一致；所诱导表达的含GST的融合rSAG2蛋白大小约43kDa，纯化后的rSAG2经SDS—PAGE电泳显示一条纯化条带；蛋白免疫印迹结果显示rSAG2能够被兔弓形虫感染血清所识别。结论在大肠埃希氏菌中融合表达了弓形虫SAG2重组蛋白，纯化的rSAG2蛋白具有一定的免疫活性。

Expression and purification of SAG2 surface antigen of Toxoplasma gondii

Objective To express and purify the surface antigen 2 （SAG2）of Toxoplasma gondii. Methods The SAG2 encoding gene was amplified by PCR,and ligated with the pMD-18T plasmid to construct a recombinant for sequencing,and then the SAG2 gene was subcloned into the plasmid pGEX-4T-2,which was expressed in E.coli JM109 in order to obtain the recombinant SAG2（rSAG2）.The rSAG2 protein was purified using GST fusion protein purification kit, and identified by SDS-PAGE and west-blotting using rabbit serum infected with T. gondii. Results The SAG2 gene fragment was amplified from the DNA of T. gondii,about 469bp in length, the sequence was the same with the homology sequence of T. gondii R.H strain deposited in GenBank （gi：161925）. The rSAG2 （containing GST）was about 43kDa,which could be recognized by rabbit serum infected with T.gondii. Conclusions The rSAG2 was expressed and purified,the protein was immunoreactive with the anti-T.gondii positive serum.