弓形虫不同分离株致密颗粒蛋白基因的序列分析及其在大肠埃希菌中的表达

Sequence Analysis and Expression of GRA7 Gene of Toxoplasma gondii Isolates in Escherichia coli

目的分析弓形虫不同分离株致密颗粒蛋白7(densegranuleprotein,GRA7)基因的异同及在大肠埃希菌中表达致密颗粒蛋白。方法从弓形虫不同分离株(RH株、ZS2株和GT株)的基因组中特异地扩增出GRA7基因,将目的基因定向克隆至原核表达载体pGEX-4T-1,转化大肠埃希菌JM109并测序。利用互联网上的在线工具CLUSTALW进行序列分析。异丙基-B-D-硫代半乳糖苷(IPTG)体外诱导pGEX-4T-1/GRA7重组质粒菌的表达,对表达的目的蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),分别以抗抗谷胱甘肽巯基转移酶(GST)抗体、人抗弓形虫阳性血清为一抗进行Westernblotting分析。以纯化的重组蛋白作为包被抗原,ELISA法检测抗弓形虫阴性、阳性血清。结果弓形虫不同分离株GRA7的基因序列相同;pGEX-4T-1/GRA7重组质粒在大肠埃希菌中表达了目的蛋白,Westernblotting分析表明该蛋白为GST融合蛋白,且能被人抗弓形虫阳性血清所识别;ELISA结果表明该蛋白能与人抗弓形虫阳性血清、兔抗弓形虫阳性血清特异结合,而与抗弓形虫阴性血清无反应。结论弓形虫不同分离株GRA7基因具有高度保守性;GRA7基因在大肠埃希菌中以GST融合蛋白的形式得到表达,且该重组蛋白具有一定免疫反应性。

Objective To analyze the difference of GRA7 gene of Toxoplasma gondii different isolated strains and express GRA7 in Escherichia coli. Methods The GRA7 gene was amplified from genomes of T.gondii isolates by PCR and was cloned into pGEX-4T-1. The recombinant plasmid was transformed into JM109 and sent to be sequenced. The sequence was analyzed with CLUSTALW (an internet tool). The recombinant plasmid was induced by IPTG to express the fusion protein,which was identified by SDS-PAGE and Western blot with positive sera. The protein was purified and used as a diagnostic antigen for ELISA to test serum samples. Results There was no difference among the sequences of T.gondii GRA7 gene from different isolates. The recombinant plasmid pGEX-4T-1/GRA7 induced by IPTG was expressed in E.coli. It was a GST fusion protein and could react with human and rabbit positive sera analyzed by Western blot. Conclusion The GRA7 gene of T.gondii isolates is highly conservative. The GRA7 is expressed as a recombinant protein in Escherichia coli, which shows an immunoreactivity.