鸡α-干扰素基因克隆及其在大肠杆菌中表达

Cloning of Chicken IFN-α Gene and Its Expression in Escherichia coli

根据GenBank中鸡IFN-α基因设计引物对,利用PCR技术克隆并测定了SPF鸡的IFN-α基因,再在成熟肽基因两侧设计一对引物,分别加入合适的酶切位点,将其亚克隆在表达载体pET上,转化BL21(DE3)后用IPTG诱导表达,用SDS-PAGE电泳分析表达形式,结果为蛋白以包涵体形式表达。提取的包涵体用7mol/L盐酸胍变性,利用胱氨酸—半胱氨酸再氧化法对包涵体变性液进行分段稀释法复性。细胞病变抑制法(CEF-VSV为基本检测系统)测定复性液的抗病毒活性不低于107.0U/mL。

According to the sequence of chicken IFN-α gene published in the GenBank, the chicken IFN-α gene was cloned and sequenced, a pair of primers were designed and synthetized which can amplify the mature peptide gene, and added to the appropriate enzyme restriction site, then the gene fragment was subcloned with vector pET, the recombinant vector was introduced into BL21 （DE3）, and analysised by SDS-PAGE after induction with IPTG. The results indicated that the protein was expressed as inclusion body. The inclusion body was dissolved in 7mol/L guandine chloride and subsequently re-natured by diluting with refolding buffer containing cystine-cysteine.The re-natured product was verified to be anti-virus activity over 10^7.0 U/mL by CPE inhibition test.