红色原鸡IFN-γ基因的克隆及原核表达  

Cloning and Prokaryotic Expression of IFN-γGene of Red Jungle Fowl

作　　者：孙亭亭[1] 马志亮[1] 冀斌[1] 胡文丽[1] 陈培富[1] 

机构地区：[1]云南农业大学动物科学技术学院,云南昆明650201

出　　处：《动物医学进展》2014年第7期47-51,共5页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31060130)

摘　　要：应用能够选择性克隆同源双链DNA的方法(PCR+1)从刀豆蛋白A诱导的红色原鸡外周血淋巴细胞中扩增γ干扰素(IFN-γ)编码区基因,双酶切后连接至克隆载体pUC18。以阳性质粒为模板扩增IFN-γ成熟肽基因,连接至原核表达载体pProEXTMHTb,筛选出阳性重组质粒HTb-IFN-γ;重组质粒在大肠埃希菌BL21(DE3)中经IPTG诱导表达,表达产物通过SDS-PAGE和Western blot进行分析。结果表明,采用PCR+1方法扩增得到560bp IFN-γ编码区基因,成功连接至克隆载体并以此为模板扩增出473bp成熟肽基因,构建的重组表达质粒HTb-IFN-γ在BL21(DE3)中经IPTG诱导表达出分子质量约为21ku的IFN-γ。Western blot分析显示,表达的IFN-γ能够与抗IFN-γ多克隆抗体特异性反应,具有良好的免疫活性。A method which permitted the selective cloning of homoduplex of molecules was used to clone the IFN-γcomplete gene from PBMC induced by Con A.The PCR＋1products were ligated with pUC18 vector and the mature peptide gene was amplified.The recombinant expression plasmid named HTb-IFN-γwas constructed and induced with IPTG in E.coli BL21（DE3）.The expression products were analyzed by SDS-PAGE and Western blot.The results showed that a fragment containing 560 bp was amplified by PCR＋1and mature peptide gene containing 473 bp was obtained from positive plasmid pUC-IFN-γ.SDSPAGE and Western blot analysis indicated that induced with IPTG in E.coli BL21（DE3）,IFN-γwas expressed with the size about 21 ku and could be combined with polyclonal antibody against IFN-γwhich made clear that the products had immunological reactive activity.

关 键 词：红色原鸡 IFN-Γ 不对称PCR 原核表达 

分 类 号：S852.43[农业科学—基础兽医学] Q786[农业科学—兽医学]