CAJ | 학술논문

目的：构建弓形虫Chinese 1基因型TgCtWh3（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pMD18-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pET-28a和真核表达载体pEGFP-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3 D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pET-28a和真核表达载体pEGFP-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pET-28a和pEGFP-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。
목적：구건궁형충Chinese 1기인형TgCtWh3（이후균간칭Wh3）주봉상체단백（ rhoptry protein ，ROPs）16적원핵화진핵중조표체질립급기3D결구。방법삼조ROP16서렬분별설계인물，채용PCR종궁형충Chinese 1기인형Wh3주기인조DNA중확증출편마ROP16적기인편단，극륭지pMD18-T재체；경PCR급측서분석감정；양성극륭적질립분별아극륭지원핵표체재체pET-28a화진핵표체재체pEGFP-C2，분별전화대장간균BL21화DH5α，PCR화매절감정전화균락삽입적서렬；장구건적원핵표체균주경IPTG유도，SDS-PAGE화면역인적분석융합단백적표체；장구건적진핵중조질립경지질체전염293T세포，관찰기재세포중표체；채용생물신식학방법분석구건료단백적3 D결구。결과각조균PCR확증출약2．1 kb ROP16기인적특이편단，서렬검측결과균정학；분별아극륭도원핵표체재체pET-28a화진핵표체재체pEGFP-C2중，성공구건료Chinese 1기인형궁형충봉상체단백ROP16적원핵표체질립화진핵표체질립；원핵표체질립재대장간균중표체료ROP16적융합단백；진핵표체질립재293T세포중성공표체，성공구건출ROP16기인적3D결구도。결론이pET-28a화pEGFP-C2위재체，분별성공구건병표체료ROP16적원핵화진핵중조질립，병구건출기3D결구도。
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.