刚地弓形虫滑行相关蛋白45的克隆、表达与免疫反应性检测

Cloning, expression and immunoreactivity of Toxoplasma gondii gliding-associated protein 45

目的克隆、原核表达刚地弓形虫滑行相关蛋白45(TgGAP45)基因,检测重组蛋白rTgGAP45的免疫反应性。方法提取刚地弓形虫RH株速殖子总RNA,逆转录合成cDNA。根据TgGAP45基因全长编码序列的开放阅读框设计引物,PCR扩增TgGAP45基因。扩增产物经双酶切后连接入pET30a(+)载体,重组质粒转化至大肠埃希菌(E.coli)DH5α,阳性菌落经PCR、双酶切和测序鉴定。将重组质粒pET-30a(+)-TgGAP45转化至E.coli BL21(DE3)感受态细胞,以异丙基-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,分别以抗His标签抗体、人抗弓形虫血清或小鼠抗弓形虫可溶性速殖子抗原(STAg)血清为一抗,蛋白质印迹(Western blotting)鉴定重组蛋白免疫反应性。结果刚地弓形虫GAP45基因的cDNA开放式阅读框全长为738 bp,PCR扩增结果显示,在750 bp处有一条特异性扩增条带(带His标签),与预期大小相符。菌落PCR、双酶切以及测序结果均表明重组质粒pET-30a(+)-TgGAP45构建成功。SDS-PAGE结果表明,经IPTG诱导获得相对分子质量(Mr)约35000的可溶性重组蛋白。Western blotting分析结果显示,诱导表达的蛋白为带His标签的重组蛋白,可分别被His标签抗体、人抗弓形虫血清和小鼠抗弓形虫STAg血清识别。结论刚地弓形虫RH株TgGAP45基因片段可在原核表达系统中高效可溶性表达,该重组蛋白rTgGAP45具有免疫反应性。

Objective To clone and express Toxoplasma gondii gliding-associated protein 45(TgGAP45)as recombinant protein and analyze its immunoreactivity by T.gondii infected human serum for its potential as an immunodiagnostic antigen.Methods Total RNA was extracted from tachyzoites of T.gondii RH strain and reversely transcribed into cDNA(RT-PCR).The DNA encoding for the open reading frame of TgGAP45 was amplified with gene-specific primers based on TgGAP45 sequence(GenBank accession No.AF453384.1).The PCR products were cloned into bacterial expression vector pET-30a(+)using EcoRⅠand XhoⅠsites.The correct recombinant plasmid pET-30a(+)-TgGAP45 was identified by PCR with gene-specific primers,double restriction enzyme digestion and DNA sequencing.The pET-30a(+)-TgGAP45 plasmid DNA was transformed into E.coli Rosetta(DE3)and the recombinant TgGAP45 protein(rTgGAP45)was expressed under induction of IPTG.The rTgGAP45 was purified with Ni-NTA column and analyzed by SDS-PAGE for its purity and Western blotting for its immune-recognition by T.gondii infected human serum and mouse anti-T.gondii soluble tachyzoites antigen(STAg)serum.Results DNA coding for TgGAP45 was successfully amplified from T.gondii tachyzoites total RNA by RT-PCR as 750 bp in length,then cloned into bacterial expression vector pET-30a(+).The correct recombinant plasmid pET-30a(+)-TgGAP45 was identified by gene-specific PCR,double restrict enzyme digestion and DNA sequencing.The TgGAP45 was successfully expressed as soluble recombinant protein in E.coli BL21(DE3)under induction of IPTG with a rough molecular weight of 35000,and purified with Ni-NTA.The His-tagged rTgGAP45 was strongly recognized not only by the anti-His antibody,but also by T.gondii-infected human serum and mouse anti-T.gondii STAg serum by Western blotting.Conclusion DNA encoding for TgGAP45 was successfully cloned from T.gondii tachyzoites and rTgGAP45 was efficiently expressed as soluble protein in E.coli BL21(DE3).The purified rTgGAP45 remained its immunoreactivity to T.gondii infected human serum and mouse immune sera.