刚地弓形虫磷酸甘油酸变位酶2基因片段克隆、表达及抗原性分析

目的克隆、表达刚地弓形虫(Toxoplasma gondii)磷酸甘油酸变位酶2(TgPGAM2)基因片段,并分析其抗原性。方法提取弓形虫RH株速殖子总RNA,逆转录合成cDNA。PCR扩增TgPGAM2基因。扩增产物经双酶切后连接入pET30a(+)载体,重组质粒转化大肠埃希菌(E.coli)DH5α,阳性菌落经PCR和双酶切鉴定,并测序。将测序正确的重组质粒pET30a(+)-TgPGAM2转化至E.coli BL21并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物。以兔抗弓形虫血清为一抗,蛋白质印迹(Western blotting)分析重组蛋白的抗原性。结果PCR扩增产物约为750 bp。菌落PCR、双酶切和测序结果显示,重组质粒pET30a(+)-TgPGAM2构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量(Mr)约30 000的可溶性重组蛋白。Western blotting分析证实其能被兔抗弓形虫血清识别。结论刚地弓形虫RH株TgPGAM2基因片段可在原核表达系统中表达,且该可溶性重组蛋白具有抗原性。

Cloning, expression and antigenicity analysis of phosphoglycerate mutase 2 gene of Toxoplasma gondii

Objective To clone and express the phosphoglycerate mutase 2(PGAM2)gene of Toxoplasma gondii,and analyze the antigenicity of the recombinant protein.Methods Total RNA was extracted from T.gondii tachyzoites of RH strain and reversely transcribed into cDNA.TgPGAM2 gene was amplified by PCR and cloned into pET30a(+)vector.The constructed pET30a(+)-TgPGAM2 was transformed into E.coli DH5α first and selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing.The correct plasmid was transformed into E.coli BL21 for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by Coomassie brilliant blue staining.Western blotting assay with rabbit anti-T.gondii serum was used to analyze its antigenicity.Results The length of PCR product was about 750 bp and the recombinant plasmid pET30a(+)-TgPGAM2 was successfully constructed.The results of SDS-PAGE and Western blotting revealed that the relative molecular weight(Mr)of the soluble recombinant protein was approximately 30 000 and could be recognized by rabbit anti-T.gondii serum.Conclusion The soluble TgPGAM2 protein has been expressed in the prokaryotic expression system and maintains its antigenicity.