Chinese1基因型弓形虫棒状体蛋白ROP16的基因克隆表达及生物信息学分析

Gene cloning and bioinformatics analysis of gene ROP16 of Toxoplasma Gondii with Genotype Chinese

目的构建弓形虫Chinese 1基因型Tg Ct Wh3(以后均简称Wh3)株棒状体蛋白(rhoptry protein,ROPs)16的原核和真核重组表达质粒及其3D结构。方法参照ROP16序列分别设计引物,采用PCR从弓形虫Chinese 1基因型Wh3株基因组DNA中扩增出编码ROP16的基因片段,克隆至p MD18-T载体;经PCR及测序分析鉴定;阳性克隆的质粒分别亚克隆至原核表达载体p ET-28a和真核表达载体p EGFP-C2,分别转化大肠杆菌BL21和DH5α,PCR和酶切鉴定转化菌落插入的序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE和免疫印迹分析融合蛋白的表达;将构建的真核重组质粒经脂质体转染293T细胞,观察其在细胞中表达;采用生物信息学方法分析构建了蛋白的3D结构。结果各组均PCR扩增出约2.1 kb ROP16基因的特异片段,序列检测结果均正确;分别亚克隆到原核表达载体p ET-28a和真核表达载体p EGFP-C2中,成功构建了Chinese 1基因型弓形虫棒状体蛋白ROP16的原核表达质粒和真核表达质粒;原核表达质粒在大肠杆菌中表达了ROP16的融合蛋白;真核表达质粒在293T细胞中成功表达,成功构建出ROP16基因的3D结构图。结论以p ET-28a和p EGFP-C2为载体,分别成功构建并表达了ROP16的原核和真核重组质粒,并构建出其3D结构图。

Objective construct the Toxoplasma Gondii with Genotype Chinese 1 Wh3 strain rhoptry protein ROP16 in prokaryotic and eukaryoticrecombinant expression plasmid and the structure of 3D .Methods according to the sequences of ROP16 primers were de-signed,from the Chinese 1 gene of Toxoplasma gondiiWh3 strain genome DNA were amplified by ROP16 gene encoded by PCR,cloned into pMD18-T vector by PCR and sequencing analysis;identification;plasmid groups of positive clones were subcloned into prokaryotic expression vector pET-28a and the eukaryotic expression vector pEGFP-C2,BL21 and DN5a were transformed into Escherichia coli,se-quences of PCR and enzyme digestion identification of transformed colonies inserted;the constructedprokaryotic expression was induced by IPTG,expression of the fusion proteinof SDS-PAGE and Western blot;the constructed recombinant plasmidtransfected 293T cells, and observe its expression in cells;construct the 3D structural proteins using bioinformatics analysis.Results The groups werePCR amplified specific fragments of about 2.1 kb ROP16 gene,sequence detection results are correct;they were subcloned into prokaryotic expression vector pET-28a and the eukaryotic expression vector pEGFP-C2,to constructthe prokaryotic expression plasmid and the eu-karyotic expression plasmids of Chinese 1 gene of Toxoplasma gondii rhoptry protein ROP16;prokaryotic expression the plasmid was ex-pressed in Escherichia coli ROP16 fusion protein;eukaryotic expression plasmid was successfully expressed in 293T cells;Construction of the 3D structure chart of ROP16 gene successfully.Conclusion using pET-28a and pEGFP-C2 as the carrier,respectively,to con-struct ROP16 prokaryotic and eukaryotic recombinant expression plasmid;construction of the 3D structure chart of ROP16 gene.