重组弓形虫GRA8的表达及其诱导的免疫应答

目的表达和纯化弓形虫RH株GRA8的截短型片段,分析其诱导的免疫应答。方法从重组克隆pMD18-GRA8中切取GRA8的插入片段,亚克隆至原核表达质粒pET-23a(+)中,转化大肠杆菌BL21,PCR和酶切鉴定转化菌落的插入序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE分析重组蛋白的表达;大量诱导表达GRA8,金属螯合层析予以纯化,将纯化蛋白免疫小鼠,观察其诱导的免疫应答。结果GRA8基因的特异片段被亚克隆到原核表达质粒pET-23a(+),在大肠杆菌中未见GRA8的明显表达;表达的蛋白经金属螯合层析获得纯化;纯化蛋白能被弓形虫感染兔血清识别。结论成功构建了GRA8的原核重组表达质粒,纯化的蛋白具有良好的抗原性。

Expression of the recombinant dense granule protein of Toxoplasma gondii and its induction of immune responses

The truncated fragment of Toxoplasma gondii strain RH gene encoding the dense granule antigen 8(DGA8) was expressed and the induction of immune responses with this recombinant protein were investigation in the present study,in which the right gene fragment encoding GRA8 in pMD18-GRA8 was digested with restrictive endonucleases,and was subcloned into pET32a(+).Then,the recombinant plasmid pET-23a(+)-GRA8 was transformed to E.coli BL21.and the recombinant clone was characterized by PCR and digestion with restrictive endonucleases.Meanwhile,the positive clone was induced with IGTP to express the target protein,and the expressed protein was characterized by SDS-PAGE.The recombinant protein was purified from E.coli lysates by metal-chelating chromatography,and the immune activities of this protein was subjected to analysis with immunoblotting assay and induction of immune responses in mice.The experimental results showed that the prokaryotic expression plasmid pET23a(+)-GRA 8 was constructed through subcloning the right insert of GRA8 into pET23a(+).The expression of the recombinant protein expressed in clones containing pET23a(+)-GRA8 was not so obvious when it was induced by IGTP.Meanwhile,the expressed recombinant protein was purified by metal-chelating chromatography,and the purified protein reacted with the sera from rabbits infected with T.gondii RH strain,and could induce moderate degree of immune responses.It is concluded that the prokaryotic expression plasmid pET23a(+)-GRA8 was successfully constructed and the purified protein shows good antigenicity.