弓形虫RH株致密颗粒蛋白GRA4基因的克隆与表达

目的 克隆和表达弓形虫RH株致密颗粒蛋白GRA4基因。方法 根据GRA4基因序列,设计合成一对引物,用聚合酶链式反应(PCR)方法从弓形虫RH株基因组DNA中扩增GRA4基因片段,插入pMD18-T载体,并转化大肠杆菌JM109,经PCR、双酶切、测序验证后,将GRA4基因片段定向亚克隆到载体pGEX-4T-2中构建原核表达重组质粒pGEX-4T-2.GRA4,重组子在E.coli BL21中经IPTG诱导表达,并对表达产物进行SDS-PAGE及Westem blot分析。结果 从弓形虫RH株基因组DNA中扩增出GRA4基因片段并诱导表达出能被兔抗弓形虫血清识别的重组GRA4蛋白。结论 成功构建和表达了弓形虫pGEX-4T-2-GRA4重组质粒,为弓形虫病诊断抗原和疫苗的研究奠定了基础。

Cloning and expression of GRA4 gene of Toxoplasma gondii RH strain

To done and express the gene coding dense granule protein 4 (GRA4) of Toxoplasma gondii RH strain, a pair of primers were designed and synthesized, according to the sequence of GRA4 gene, by using PCR technique, and the specific fragment of GRA4 gene was obtained by amplification of genomic DNA of Toxoplasma gondii RH strain. After purification, the fragments of PCR products were cloned into pMD18-T vector, then transferred into E. coli JM109. Positive clones were identified by PCR, restrictive enzyme digestion and sequence analysis. The inserted GRA4 gene was subcloned into pGEX-4T-2 vector, a recombinant pGEX-4T-2-GRA4 was constructed. The fusion recombinant protein was expressed in E. coli BL21 and was analyzed by means of SDS-PAGE and Western blotting.The specific gene fragment GRA4 was amplified form genomic DNA of Toxoplasma gondii RH strain, and the recombinant plasmid pGEX-4T-2-GRA4 could express a fusion protein in E. coli BL21 which could be recognized by immune rabbit serum.The recombinant plasmid which contain the gene fragment encoding GRA4 of T. gondii has been successfully constructed and expressed.This provides the basis for the diagnosis and vaccination against Toxoplasmosis.