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

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.