刚地弓形虫ROP10-ROP18真核表达载体的构建与鉴定  

Construction and identification of a dual promoter eukaryotic expression vector for ROP10 and ROP18 of Toxoplasma gondii

作　　者：刘荣荣[1] 张晓磊[1] 张进顺[1] 贾晓晖[1] 刘楠[1] LIU Rong-rong;ZHANG Xiao-lei;ZHANG Jin-shun;JIA Xiao-hui;LIU Nan(Institute of Pathogen Biology 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》2018年第6期612-616,共5页Journal of Pathogen Biology

基　　金：河北省自然科学基金项目(No.H2013405091);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10(ROP10)、棒状体蛋白18(ROP18)复合基因真核表达载体pVAXDROP10-ROP18,并在HeLa细胞内表达目的蛋白。方法设计ROP10、ROP18基因特异引物,采用RT-PCR扩增ROP10、ROP18基因并测序,将ROP10和ROP18基因分别定向插入双启动子真核表达载体pVAXD的多克隆位点中,构建单价质粒pVAXD-ROP10和pVAXD-ROP18,然后将ROP18基因插入pVAXD-ROP10中构建双启动子真核表达载体pVAXD-ROP10-ROP18。经PCR和双酶切验证后将pVAXD-ROP10-ROP18转染至HeLa细胞内,提取细胞总RNA并逆转录为cDNA,以此为模板分别进行ROP10基因和ROP18基因的RT-PCR鉴定;采用间接免疫荧光试验(IFA)检验ROP10和ROP18蛋白表达情况。结果成功克隆ROP10、ROP18基因片段并构建了重组质粒pVAXDROP10-ROP18,测序、双酶切和PCR验证重组质粒构建正确。分别将3种重组质粒转染至HeLa细胞后经RT-PCR鉴定,pVAXD-ROP10-ROP18组可同时扩增出1 761bp(ROP10基因)和1 665bp(ROP18基因)2个片段,而pVAXDROP10组和pVAXD-ROP18组分别扩增出1 761bp和1 665bp的目的片段;IFA显示pVAXD-ROP10组和pVAXDROP18组均有绿色荧光,即分别表达ROP10和ROP18蛋白。结论成功构建重组质粒pVAXD-ROP10-ROP18、pVAXD-ROP10和pVAXD-ROP18质粒可在真核细胞中分别表达ROP10和ROP18两种蛋白。Objectives To construct a dual promoter eukaryotic expression vector（pVAXD-ROP10-ROP18）for rhoptry protein 10（ROP10）and rhoptry protein 18（ROP18）of Toxoplasma gondii and to verify its expression in HeLa cells. Methods Gene fragments of ROP10 and ROP18 were amplified using RT-PCR and sequenced.The ROP10 gene and the ROP18 gene were respectively inserted into the multiple cloning sites of the dual promoter eukaryotic expression vector pVAXD to construct the plasmids pVAXD-ROP10 and pVAXD-ROP18.The ROP18 gene was inserted into the plasmid pVAXD-ROP10 to construct the dual promoter eukaryotic plasmid pVAXD-ROP10-ROP18.Double enzyme digestion and PCR verified that the recombinant plasmid pVAXD-ROP10-ROP18 was correctly constructed.The plasmid was then transfected into HeLa cells.Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA,which was used as a template to amplify the ROP10 gene and the ROP18 gene.An indirect immunofluorescence assay（IFA）was performed to determine the expression of each gene. Results Gene fragments of ROP10 and ROP18 were successfully cloned,and the recombinant plasmid pVAXD-ROP10-ROP18 was constructed.After the plasmid was transfected into HeLa cells,RT-PCR indicated that pVAXD-ROP10-ROP18 simultaneously produced bands of 1 761 bp and 1 665 bp,while pVAXD-ROP10 produced bands of 1 761 bp and 1 665 bp.In the IFA,green fluorescence was observed in HeLa cells transfected with pVAXD-ROP10 and pVAXD-ROP18. Conclusion The recombinant plasmid pVAXD-ROP10-ROP18 was successfully constructed and expressed in eukaryotic cells.

关 键 词：刚地弓形虫 棒状体蛋白10 棒状体蛋白18 重组质粒 真核表达载体 

分 类 号：R382.5[医药卫生—医学寄生虫学]