鸡干扰素α1及胸腺肽α1融合蛋白原核表达系统的建立

Establishment of prokaryotic expression system of chicken IFNα1-THYα1 fusion protein

根据GenBank已发表的鸡干扰素α1(IFNα1)基因序列,设计1对特异性引物,采用PCR方法,从鸡基因组中扩增出鸡IFNα1基因。根据胸腺肽α1(THYα1)氨基酸序列,选用大肠杆菌偏爱密码子,设计基因序列,同时在IFNα1-THYα1之间设计1个5肽Linker。在IFNα1基因结构基础上,设计4条寡核苷酸单链作为下游引物,以搭桥的方式延伸和扩增,最终获得IFNα1-THYα1融合基因;将该基因克隆至pGEX4T-1载体后,转化至BL21(DE3)受体菌中,37℃培养至菌液D600为0.6～1.0时,IPTG(1.0mmol/L)诱导培养4h;经western blot鉴定证实,成功构建了鸡干扰素α1及胸腺肽α1融合蛋白原核表达系统,表达产物以包涵体形式存在,获得高纯度的IFNα1-THYα1蛋白。以细胞病变抑制试验及E玫瑰花结形成试验对目的蛋白进行体外活性检测,结果显示该蛋白具有生物活性。

Accoding to the published genic sequences of chicken interferon-α1 ,a pair of specific primers was de signed to amplified the genc of chicken interferon-α1 by polymerase chain reaction （PCR） from the complete genome of chicken. Based on IFN α1 amino acid sequence and the choice of E. coli preference for the codon, we designed a linker of 5 peptides between IFN aland THY α1 and four oligonucleotide single chains as downszream primers. The IFN α1-THY α1 fusion gene was ohlained by PCR. The fusion gene was cloned into pGEX1T -1 expression plasmid and then transformed into BL21（DE3）,cultured in liquid LB at 37℃ and induced with IPTG for 4 hours while its D600 got up to 0.6-1.0. Western blot results indicated that the chicken IFNα1 -THYα1 fusion protein have been successfully expressed and the product of expression exists as the form of inclusion. The high-purity of IFNα1 THYα1 protein was obtained and the activity in vitro of the purposed protein was detected with the cytopathogenic effect inhibition test and E rosette forming assay. The results proved that the protein possess the biological activity.