小鼠FGFR1/MIP3a-Fc融合基因真核表达载体的构建及表达  

Construction and expression of eukaryotic expression plasmid vector FGFR1/MIP3a-Fc fusion gene in vitro of mice

作　　者：黄用豪[1] 和永壮 张晓钿[1] 方丹丹[1,2] 陈艳[1,2] 刘思汝[1,2] 许仙花[1] 郑少江[1,2] 

机构地区：[1]海南医学院热带病转化医学教育部重点实验室,海南海口571199 [2]海南医学院病理学系暨第一附属医院病理科,海南海口571199

出　　处：《中国热带医学》2017年第4期340-343,共4页China Tropical Medicine

基　　金：国家自然科学基金(No.81060184;81260350;81372465;81460461);海南省国际科技合作专项(No.KJHZ2014-23);海南省高等学校科学研究重点项目(No.hnky2015ZD-13);海南省自然科学基金创新团队项目(No.2017CXTD008)

摘　　要：目的构建小鼠成纤维细胞生长因子受体1/巨噬细胞炎性蛋白3a-Fc融合基因(FGFR1/MIP3a-Fc)的真核表达质粒,并在293T细胞中的表达。方法设计合成FGFR1/MIP3a融合基因引物,用PCR方法扩增获得FGFR1/MIP3a基因片段,用内切酶消化后插入pc DNA3.1(+)/Fc(Mouse Ig G2a)质粒中,构建FGFR1/MIP3a-Fc融合基因表达质粒;经PCR鉴定、酶切鉴定以及测序鉴定后,将该融合基因表达质粒瞬时转染293T细胞,用ELISA法检测其在293T细胞的表达。结果经PCR、酶切鉴定以及测序证实,FGFR1/MIP3a-Fc融合基因表达质粒构建成功;ELISA检测FGFR1/MIP3a-Fc融合基因表达质粒能够在293T细胞中表达。结论 FGFR1/MIP3a-Fc融合基因表达质粒构建成功,该质粒能够在293T细胞中表达。Objective To construct and express a eukaryotic expression plasmid of mice FGFR1/MIP3a-Fc fusion gene,and detect its expression in the cells of 293 T. Methods The FGFR1/MIP3 a fusion gene fragment was amplified by PCR with primers; After digesting by restriction endonuclease, the FGFR1/MIP3 a fusion gene fragment was inserted into the eukaryotic expression vector of pc DNA3.1（＋）/Fc（Mouse Ig G2a） to construct the expression plasmid of FGFR1/MIP3a-Fc; After confirming by PCR, enzyme digestion analysis, and sequencing, the recombinant plasmid of FGFR1/MIP3a-Fc was transferred into 293 T cells and the expression of the fusion gene was identified by ELISA. Results The identification of PCR and restriction endonuclease and DNA sequencing demonstrated that the recombinant plasmid FGFR1/MIP3a-Fc fusion gene was correctly constructed; the expression of FGFR1/MIP3a-Fc protein in 293 T cells was confirmed by ELISA. Conclusion The eukaryotic expression plasmid of FGFR1/MIP3a-Fc was successfully constructed, which can be expressed in 293 T cells.

关 键 词：成纤维细胞生长因子受体1 巨噬细胞炎性蛋白3a FC融合蛋白 真核表达质粒 基因表达 

分 类 号：Q782[生物学—分子生物学]