摘要
目的在大肠杆菌中高效表达牛γ-干扰素(bovine interferon-γ,BovIFN-γ),并对其生物活性进行初步鉴定。方法依据GenBank上基因序列人工合成BovIFN-γ基因,PCR方法扩增该基因,将其插入PET-28a载体构建原核表达质粒,转化大肠杆菌BL21中,在IPTG诱导下表达BovIFN-γ,并进行Western blot鉴定。Ni-NTA亲和层析法和电洗脱方法纯化表达的重组蛋白,用Western blot和商品化的BovIFN-γ检测试剂盒进行重组蛋白的抗原性检测。结果成功构建了BovIFN-γ原核表达载体PET-28a-BIFN-γ,并在大肠杆菌中高效表达,表达蛋白约占菌体总蛋白的32%,表达产物主要以可溶性形式存在于菌体裂解液上清中;重组蛋白可与BovIFN-γ单克隆抗体反应,Ni-NTA亲和层析法纯化的重组蛋白抗原活性比电洗脱方法纯化的抗原活性高。结论在大肠杆菌中成功表达了可溶性的BovIFN-γ蛋白,可与BovIFN-γ单抗发生反应,纯化的重组蛋白具有良好的反应原性。
Objective To express bovine interferon-γ（BovIFN-γ） in E.coli,and preliminarily identify its biological activity.Methods According to the gene sequence in GenBank to construct the BovIFN-γ gene,and use it as the template,to amplify the BovIFN-γ gene by polymerase chain reaction（PCR）.To insert the BovIFN-γ gene into vector PET-28a,transform it into E.coli BL21-competent cells,induce it with IPTG to express BovIFN-γ,and analyze it by Western blot.Using the NI-NTA affinity chromatography and electroelution to purify the recombination protein,and detect the protein antigenicity by commercial BovIFN-γ kit.Results The recombinant plasmid PET-28a-BIFN-γ was successfully constructed,and BovIFN-γ was efficiently expressed in E.coli.The protein expression accounted for 32% of the total soluble bacterial proteins.The expression products mainly existed in soluble form in the cell lysate supernatant,and the proteion could be discriminated by BovIFN-γmonoclonal antibody.The recombinant protein purified by NI-NTA affinity chromatography had higher antigen activity than that by electroelution.Conclusions The soluble recombinant BovIFN-γ protein is successfully expressed in E.coli,and the protein can react with BovIFN-γ monoclonal antibody.The purified recombinant BovIFN-γ protein has good antigenicity.
出处
《中国比较医学杂志》
CAS
2012年第2期38-42,共5页
Chinese Journal of Comparative Medicine
关键词
牛γ干扰素
原核表达
抗原性
大肠杆菌
Bovine interferon-γ
Prokaryotic expression
Antigenicity
E.coli
