锚定修饰的真核表达质粒pcDNA3.1(+)-rmCGβ-IgGFc-GPI的构建及鉴定  

Construction and identification of eukaryotic plasmid encoding pcDNA3.1(+)-rmCGβ-IgGFc-GPI

作　　者：许铁峰[1,2] 夏立平[1] 陈兴[2] 阎瑾琦[2] 于继云[2] 

机构地区：[1]海南医学院肿瘤研究所,海口570102 [2]军事医学科学院基础医学研究所

出　　处：《山东医药》2009年第27期16-19,共4页Shandong Medical Journal

基　　金：国家高技术研究发展计划(863)资助项目(2001AA217131);海南省教育厅资助项目(Hjkj2006-26)

摘　　要：目的为研究锚定修饰的rmCGβ-IgGFc-GPI的新功能和免疫治疗奠定基础。方法应用基因工程技术将pCI-IgGFc-GPI质粒经双酶切后,插入pcDNA3.1(+)真核表达质粒,构建重组真核表达质粒pcDNA3.1(+)-Ig-GFc-GPI,然后再插入经双酶切的恒河猴绒毛膜促性腺激素β亚基(rmCGβ亚基),构建重组真核表达质粒pcD-NA3.1(+)-rmCGβ-IgGFc-GPI,经限制内切酶酶切分析及测序鉴定正确后,用脂质体转染技术转染B16细胞,流式细胞仪技术和细胞免疫荧光法检测B16细胞中表达的IgGFc段融合蛋白。结果酶切和测序证明正确构建了真核表达质粒pcDNA3.1(+)-rmCGβ-IgGFc-GPI,流式细胞仪技术和细胞免疫荧光法证实该质粒能在B16真核细胞中正确表达目的蛋白;建立了稳定的细胞株B16/pcDNA3.1(+)-rmCGβ-IgGFc-GPI。结论锚定修饰的rmCGβ亚基的真核表达质粒pcDNA3.1(+)-rmCGβ-IgGFc-GPI成功构建,且能在B16细胞内表达;为进一步研究锚定修饰的rmCGβ-IgGFc-GPI的新功能和免疫治疗奠定了基础。Objective To lay the foundation for the study of new functions and immunotherapy of the GPI-modified macaca mulatta chorionic gonadotrophin β subunit（rmCGβ）.Methods The fragment cDNA of IgGFc-GPI encoding gene was acquired by restriction enzyme digestion from pCI-IgGFc-GPI.Then the cDNA was inserted into the eukaryotic expression vector pcDNA3.1（＋）to construct pcDNA3.1（＋）-IgGFc-GPI,and the rmCGβ was inserted into the resultant recombinant plasmid designated as pcDNA3.1（＋）-rmCGβ-IgGFc-GPI.The recombinant eukaryotic plasmid pcDNA3.1（＋）-rmCGβ-IgGFc-GPI was transfected into eukaryotic B16 cell by lipofectin transfection.IgGFc expression in B16 cell was detected by cellular immunofiuorescence test and flow cytometry.Results The recombinant eukaryotic expression plasmid pcDNA3.1（＋）-rmCGβ-IgGFc-GPI was successfully constructed and could express the interest protein correctly in the B16 cells;steady cell line of B16/pcDNA3.1（＋）-rmCGβ-IgGFc-GPI was constructed.Conclusions A recombinant eukaryotic expression plasmid pcDNA3.1（＋）-rmCGβ-IgGFc-GPI,included GPI-anchored protein and IgGFc expression can be constructed successfully and expressed in the B16 cell.This study provides a basis for further study on the new function and immunotherapy of rmCGβ-Pr＋-IgGFc-GPI.

关 键 词：锚定修饰蛋白 绒毛膜促性腺激素 Β亚基 

分 类 号：R97[医药卫生—药品]