弓形虫GRA8真核表达质粒的构建与体外表达

目的构建弓形虫致密颗粒蛋白GRA8的真核重组表达质粒。方法设计GRA8的特异引物,采用多聚酶链反应(PCR)技术从弓形虫RH株基因组DNA中扩增编码GRA8的基因片段,经克隆至pMD18-T载体后,亚克隆至真核表达载体pVAC而构建真核重组表达质粒pVAC-GRA8,转化大肠杆菌DH5α;将构建的真核重组表达质粒pVAC-GRA8转染vero细胞,分析转染vero细胞中GRA8的表达状况。结果PCR扩增出GRA8基因的特异片段,所获克隆的序列正确,并被亚克隆到真核表达载体pVAC,构建了真核重组表达质粒pVAC-GRA8;在vero细胞中获得表达。结论成功构建了GRA8的真核重组表达质粒pVAC-GRA8。

Construction and in vitro expression of Toxoplasma gondii GRA8 eukaryotic expression plasmid

To construct an eukaryotic expression plasmid containing gene encoding Toxoplasma gondii RH strain dense granule protein GRA8,gene fragment encoding GRA8 was amplified by PCR with special primers from Toxoplasma gondii genomic DNA, and was cloned into pMD18-T vector.The right gene fragment encoding GRA8 in positive clone was digested with BamHand EcoRand was subcloned into pVAC which was also digested with BamHand EcoR.Then the recombinant plasmid was transformed into E.coli DH5α.The positive recombinant clone was characterized by PCR and digestion with restriction endonucleases. pVAC-GRA8 was transfected into Africa green monkey kindey Vero cell lines and expression of GRA8 in vero cell lines were detected by RT-PCR and immunoblot with rabbits sera infected with Toxoplasma gondii.The results showed that the gene fragment encoding GRA8 was amplified by PCR from Toxoplasma gondii genomic DNA and the insert of GRA8 in positive clone was coincident with the original sequence of GRA8 gene from GenBank,The recombinant plasmid pVAC-GRA8 was contructed through subcloning the right insert of GRA8 into pVAC properly; GRA8 was expressed in Vero cells transfected with pVAC-GRA8.In conclusion the eukaryotic expression plasmid pVAC-GRA8 was successfully constructed.