摘要
目的:构建人干扰素α的高效表达质粒pEE14.1-IFN-α,并在真核细胞中验证其表达。方法:通过PCR获得人IFN-α基因,连入过渡载体pCI-GPI,然后克隆入高效表达载体pEE14.1中,构建重组表达质粒pEE14.1-IFN-α。瞬时转染293T细胞后48 h收获上清,采用ELISA,Western blot分别验证目的基因表达。结果:pEE14.1-IFN-α经酶切和测序分析,与预期设计完全一致,表明重组质粒构建成功。ELISA法检测瞬时转染细胞上清中α干扰素含量,浓度约为3.15 ng/mL,说明表达的蛋白有免疫活性,Western blot检测也显示该重组质粒在上清中分泌表达。结论:成功构建高效表达质粒pEE14.1-IFN-α,为慢性乙肝免疫治疗提供新的备选方案。
AIM： To construct the eukaryotic expression plasmid pEEl4,1-IFN-α expressing human IFN-α gene, and to detect the expression of the plasmid in eukaryotic cells. METHODS： The human IFN-α gene amplified by PCR and was linked into pCI-GPI, then inserted into the eukaryotic expression vector pEE14, 1. The recombinant plasmid was transfected into the 293T cells, the IFN-α expression was detected by ELISA and Western blotting. RESULTS： Enzyme digestion and sequence analysis showed that the bicistronic eukaryotic expression vector pEEl4.1-1FN-α was constructed successfully. The expression of plasmid was detected by ELISA, and the production of IFN-α in supernarant of transfected cells was about 3.15 ng/mL. Also, Western blotting could reveal the characteristic band of IFN-α gene. CONCLUSION： The vector is constructed successfully, which provide a new selection for HBV immunotherapy.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第4期381-383,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(31100655)
