人工合成肺孢子菌p55抗原串联基因真核表达载体的构建及表达  

Construction and identification of eukaryotic expression plasmids of synthetic TAG gene of Pneumocystis p55 antigen

作　　者：樊华[1,2] 国九英 马素丽[1] 张楠[1] 安春丽[1] 

机构地区：[1]中国医科大学基础医学院病原生物教研室,沈阳110122 [2]黑龙江省齐齐哈尔市第一医院检验科,齐齐哈尔161005

出　　处：《中国人兽共患病学报》2015年第5期399-402,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.81370189)资助~~

摘　　要：目的研究肺孢子菌p55抗原人工合成串联基因的真核表达载体构建及其表达鉴定。方法从肺孢子菌p55蛋白5个变异体中选取可能在肺孢子菌肺炎中起重要作用抗原表位,串联合成一段多表位基因,命名为TAG。将TAG双酶切后克隆到pIRES-hrGFP-1a真核表达载体,构建重组质粒pIRES-hrGFP-1a/TAG,将重组质粒转染至感受态TG-1大肠杆菌中进行扩增后,经提取纯化质粒再转染至HEK293细胞,用Western blot鉴定蛋白表达水平。结果构建的pIRES-hrGFP-1a/TAG重组质粒经酶切后测序鉴定,证明其序列正确,成功转染至HEK293细胞,Western blot检测其在HKE293细胞中能进行有效基因表达。结论本研究构建了pIRES-hrGFP-1a/TAG重组质粒,并在HEK293细胞中成功表达,为进一步阐明TAG的免疫保护功能以及核酸疫苗的研究奠定基础。We constructed eukaryotic expression plasmids of synthetic TAG gene of Pneumocystis p55 antigen to lay the foundation for the of vaccine research of nucleic Pneumocystis pneumonia.In a series of synthetic genes for the antigen gene TAG by double digestion,it was cloned in pIRES-hrGFP-1aeukaryotic expression vector to construct recombinant plasmids as pIRES-hrGFP-1a/TAG.After propagating in E.coli TG-1,the recombinant plasmids were transfected into HEK293 cells.After 48 hincubation,Western blot was performed to identify the expression of pIRES-hrGFP-1a/TAG antigenic gene.Results showed that construction of pIRES-hrGFP-1a/TAG recombinant plasmid was expressed by restriction enzyme digestion and sequencing,and the sequence was verifyed,successfully transfected into HEK293 cells.Western blot was used to test the effective gene expression in HKE293 cells.In this study,the recombinant plasmids of pIRES-hrGFP-1a/TAG are conducted successfully and expressed in the HEK293 cells,which provide a basis for clarification of immunologic function of pIRES-hrGFP-1a/TAG and study of DNA vaccine.

关 键 词：肺孢子菌 p55抗原 串联抗原基因 真核表达 

分 类 号：R392[医药卫生—免疫学]