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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.coli JM109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469 bp;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。
Objective To express Toxoplasma gondii surface antigen 2 (SAG2) in Escherichia coli and purify the recombinant protein rSAG2. Methods The fragment of SAG2 gene was amplified from the genome of Toxoplasma gondii by polymerase chain reaction (PCR) and cloned into pGEX-4T-2 vector pGD-18T. The sequence was subcloned into expression vector pGEX-4T-2 and transformed into E. coli. E. coli JM109 competent cells were induced to express rSAG2 protein by IPTG. Recombinant rSAG2 protein was purified by B-PER glutathione S-transferase (GST) fusion protein purification kit and identified by SDS-PAGE and Western-blot. Results The amplified fragment of SAG2 gene was 469 bp. The sequencing result showed that the sequence of the cloned SAG2 gene was identical with that of RH strain Toxoplasma gondii GenBank (sequence number GI: 161925). The induced GST-containing fusion The size of rSAG2 protein was about 43 kDa. The purified rSAG2 showed a purified band by SDS-PAGE electrophoresis. Western blotting showed that rSAG2 was recognized by the serum of rabbits infected with Toxoplasma gondii. Conclusion Toxoplasma gondii SAG2 recombinant protein was expressed in Escherichia coli. The purified rSAG2 protein has some immunogenicity.