pEGFP-N1-HBsAg-ROP2多基因重组表达质粒的构建及表达鉴定  

Construction and expression of multi-gene recombinant plasmid pEGFP-N1-HBsAg-ROP2

作　　者：马荣 肖婷[1,2] 李瑾[1,2] 孙慧[1,2] 徐超 黄炳成[1,2] 尹昆 赵桂华[1,2] 崔勇[1,2] 朱嵩[1,2] 刘功振[1,2] 闫歌[1,2] 魏庆宽 MA Rong;XIAO Ting;LI Jin;SUN Hui;XU Chao;HUANG Bing-cheng;YIN Kun;ZHAO Gui-hua;CUI Yong;ZHU Song;LIU Gong-zhen;YAN Ge;WEI Qing-kuan(School of Medicine and Life Sciences, University of JinanShandong Academy of Medical Sciences, Jinan 250000, China;Shandong Institute of Parasitic Diseases, Jining 272033, China;2 Jining No. 1 People＇ s Hospital, Shandong Province, Chin)

机构地区：[1]济南大学山东省医学科学院医学与生命科学学院,济南250000 [2]山东省寄生虫病防治研究所,济宁272033 [3]山东省济宁市第一人民医院

出　　处：《中国血吸虫病防治杂志》2018年第2期184-188,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(81501770); 山东省自然科学基金(ZR2015YL051、2009ZRC03083); 山东省医药卫生科技发展计划项目(2014WS0330); 山东省医科院医药卫生科技创新工程

摘　　要：目的构建p EGFP-N1-HBs Ag-ROP2重组质粒,并转染HEK293T细胞进行表达鉴定,为弓形虫病核酸疫苗的研制奠定基础。方法根据HBs Ag基因序列和pc DNA3-p30-ROP2重组质粒酶切位点设计引物,PCR扩增HBs Ag基因,经酶切、连接、转化,利用HBs Ag基因替换p30基因,构建pc DNA3-HBs Ag-ROP2重组质粒;经HindⅢ和KpnⅠ双酶切,将HBs Ag-ROP2片段与p EGFP-N1真核表达载体相连,构建p EGFP-N1-HBs Ag-ROP2重组表达质粒,转染HEK293T细胞,观察其蛋白表达水平。结果 HBs Ag片段PCR产物约700 bp,与理论值相符;构建pc DNA3-HBs Ag-ROP2重组质粒,双酶切电泳后得到约5.4 kb和1.9 kb的两条带,与预期结果相符。p EGFP-N1-HBs Ag-ROP2双酶切后产生约4.7 kb和1.9 kb的条带,经测序鉴定,与Gen Bank发表的序列同源性为99.84%。目的基因已成功转染入HEK293T细胞中,且正确表达,蛋白浓度为3.08 mg/ml。结论成功构建p EGFP-N1-HBs Ag-ROP2重组表达质粒,转染HEK293T细胞能正确表达。Objective for expression,and pay a way for Toxoplasma gondii nucleic acid vaccine development.Methods According to the HBs Ag gene sequence and pc DNA3-p30-ROP2 recombinant plasmid restriction sites,the HBs Ag gene was amplified by PCR. The HBs Ag gene was cloned into the pc DNA3-p30-ROP2 and instead of p30 gene. The HBs Ag-ROP2 fragment was amplified by PCR and digested with HindⅢ and KpnⅠ to clone into the p EGFP-N1 eukaryotic expression vector and construct the recombinant p EGFP-N1-HBs Ag-ROP2. The expression vector was transfected into HEK293 T cells based on the identification of PCR amplification,restriction endonucleases and sequencing.Results The PCR product of HBs Ag was about 700 bp,which was consistent with the theoretical value. Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pc DNA3-HBs Ag-ROP2 recombinant plasmid. The recombinant plasmid p EGFP-N1-HBs Ag-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBs Ag-ROP2 fragment. The results of sequencing showed that the sequence was 99.84% identical with the published sequence in Gen Bank. The target plasmid was successfully transfected into HEK293 T cells,and the expression was correct,the protein concentration was 3.08 mg/ml.Conclusion The recombinant plasmid p EGFP-N1-HBs Ag-ROP2 is successfully constructed and expressed efficiently.

关 键 词：弓形虫病 乙型肝炎 PEGFP-N1 乙型肝炎病毒表面抗原(HBsAg) 弓形虫棒状体蛋白2(ROP2) 质粒构建 

分 类 号：R531.8[医药卫生—内科学]