摘要
目的在大肠杆菌中高效表达组氨酸标签与人β干扰素融合蛋白,并进行蛋白纯化和生物活性测定。方法提取人Bel-7402细胞总DNA为模板,经PCR获得人β干扰素编码基因片段。将此基因插入表达载体pET-16b,重组克隆,经酶切与测序确认后转化至大肠杆菌BL21(DE3),进行IPTG诱导表达和条件优化。表达产物经Western blotting分析,用Ni sepharose亲和层析纯化,纯化产物进行干扰素生物学活性测定。结果经表达条件优化后,SDS-PAGE显示重组菌株表达与组氨酸标签人β干扰素分子量大小一致的条带,进一步的Western blotting分析中发现表达的蛋白能与人β干扰素抗体及组氨酸标签抗体产生结合反应,亲和层析纯化分离得到纯度92%的组氨酸标签人β干扰素,干扰素生物学活性测定表明,获得的组氨酸标签人β干扰素比活性为4.2×107U/mg。结论组氨酸标签人β干扰素编码基因在大肠杆菌中成功表达。建立了该蛋白的简便亲和纯化方法,表达产物具有人β干扰素生物学活性,为进一步以此系统表达及纯化其他具有生物学活性的科研用标签化细胞因子奠定了基础。
Objective To express and purify the fusion protein HIS-IFNβ of human interferon β（IFNβ） and His-tag in E.coli BL21（DE3）.Methods IFNβ gene was obtained from the total DNA of human Bel-7402 cell by PCR.IFNβ gene was then inserted into prokaryotic expression vector pET-16b and transformed into E.coli BL21（DE3）.The expressed product was purified by affinity chromatography using Ni-NTA column and its antigenicity was analyzed by Western blotting analysis.The specific biological activity was detected by standard survival activity test on Wish cells challenged with VSV virus.Results SDS-PAGE result indicated HIS-IFNβ was expressed at the right molecular weight after induced by IPTG.Western blotting test showed the protein had reaction with either IFNβ monoclonal antibody or His-tag antibody.The purity of HIS-IFNβ purified by affinity chromatography reached 92% and the specific activity was about 4.2 × 107 U/mg.Conclusions The fusion protein of human interferon β with His-tag was successfully expressed and purified in E.coli BL21（DE3）.The bioactivity of the fusion protein has been identified by cell anti-virus test.
出处
《中国医药生物技术》
CSCD
2011年第1期12-17,共6页
Chinese Medicinal Biotechnology
基金
国家科技支撑计划(2006BAF07B01
2009BAK61B04)
国家自然科学基金(30973668)
北京市自然科学基金(5102032)
关键词
干扰素Β
重组融合蛋白质类
基因表达
Interferon-beta
Recombinant fusion proteins
Gene expression
