刚地弓形虫ROP16-ROP18复合基因真核表达载体的构建  

Construction of a eukaryotic expression vector containing the ROP16 and ROP18 genes of Toxoplasma gondii

作　　者：刘楠[1] 张晓磊[1] 郭玲玲[1] 张进顺[1] 贾晓晖[1] 刘荣荣[1] LIU Nan ZHANG Xiao-lei GUO Ling-ling ZHANG Jin-shun JIA Xiaohui LIU Rong-rong(Institute of Pathogenic Microbes and Immunology, Hebei North University, Zhangjiakou 075000, Chin)

机构地区：[1]河北北方学院病原生物学与免疫学研究所,河北张家口075000

出　　处：《中国病原生物学杂志》2017年第1期57-61,共5页Journal of Pathogen Biology

基　　金：河北省自然科学基金项目(No.2013405091);河北北方学院校级重大课题(No.ZD201312)

摘　　要：目的构建刚地弓形虫棒状体蛋白16(TgROP16)、棒状体蛋白18(TgROP18)复合基因真核表达重组质粒,并验证其在真核细胞中的表达情况。方法重组质粒pVAX1-ROP16与pVAX1-ROP18分别经EcoRⅠ和NotⅠ、NheⅠ和AflⅡ双酶切,将ROP16与ROP18基因先后克隆至双启动子真核表达载体质粒pVAXD的多克隆位点中,构建pVAXD-ROP16-ROP18重组质粒。分别用双酶切和PCR验证正确的重组质粒及对照组质粒转染Hela细胞,提取细胞总RNA并逆转录为cDNA,以此为模板进行ROP16基因、ROP18基因与管家基因β肌动蛋白基因的RT-PCR扩增,采用间接免疫荧光法检测各自基因的表达。结果重组质粒pVAXD-ROP16-ROP18经PCR及双酶切鉴定构建正确。转染后经RT-PCR检测,实验组与对照组β-肌动蛋白基因扩增产物均与预期大小相符,实验组ROP16基因、ROP18基因扩增产物分别为1 700bp和2 100bp,与预期大小相符,而对照组均未扩增出ROP16及ROP18基因。间接免疫荧光法检测显示,重组质粒pVAXD-ROP16、pVAXD-ROP18转染组细胞可见绿色荧光,空质粒及对照组无绿色荧光。结论成功构建了重组质粒pVAXD-ROP16-ROP18,该质粒可在真核细胞中表达。Objectives To construct a multicomponent eukaryotic expression plasmid containing the ROP18 gene and the ROP16 gene of Toxoplasma gondii and to examine its expression in eukaryotic cells. Method The recombinant plasmid pVAX1-ROP16 was digested with EcoRI and NotI and the recombinant plasmid was digested with NheI,and AflII.The ROP16 and ROP18genes were cloned into multiple cloning sites of the dual-promoter eukaryotic vector pVAXD to construct the recombinant plasmid pVAXD-ROP16-ROP18.After digestion with enzymes and amplification with PCR indicated that the plasmid was correct,the recombinant plasmid and the control group plasmid were transfected into Hela cells.cDNA was reverse-transcribed from RNA extracted from cultured cells.cDNA was used as a template to amplify the ROP16 gene,the ROP18 gene,and the housekeeping geneβ-actin,and indirect immunofluorescence was used to determine the expression of each gene. Results The recombinant plasmid pVAXD-ROP16-ROP18 was verified as correct with PCR and double restriction enzyme digestion.After transfection,RT-PCR indicated that the product of amplification of the beta-actin gene in the experimental group and the control group was consistent with the expected size.Products of amplification of the ROP16 gene and the ROP18 gene in the experimental group were consistent with the expected size.The product of amplification of the ROP16 gene was about 1,700 bp in length and that of the ROP18 gene was about 2100 bp in length.The ROP16 and ROP18genes were unable to be amplified in the control group.An indirect immunofluorescence test indicated that cells transfected with the recombinant plasmid pVAXD-ROP16 or pVAXD-ROP18 fluoresced green.Cells transfected with the empty plasmid transfected and the control group did not fluoresce green. ConclusionThe recombinant plasmid pVAXD-ROP16-ROP18 was successfully constructed and it was successfully expressed in eukaryotic cells.

关 键 词：刚地弓形虫 棒状体蛋白16 棒状体蛋白18 复合重组质粒 真核表达 

分 类 号：R382.5[医药卫生—医学寄生虫学]