日本大耳白兔干扰素-γ基因的克隆及其在大肠杆菌中的表达

Clone and Expression of IFN-γ Gene of Japanese White Rabbit in E.coli

提取经ConA刺激后的日本大耳白兔外周血淋巴细胞总RNA,应用RT-PCR技术对其IFN-γ基因进行扩增和测序。将去除信号肽的IFN-γ基因片段插入原核表达载体pET-30a并在大肠杆菌中进行表达。结果显示,克隆的IFN-γ基因长为504个核苷酸,具有一个完整的开放阅读框,编码168个氨基酸,所测序列与GenBank中已登录的安哥拉兔、中国白兔、欧洲兔、新西兰兔IFN-γ同源性为100%,与獭兔同源性高达99.8%。构建的重组菌经过IPTG诱导后,通过SDS-PAGE电泳证实,IFN-γ基因片段在大肠杆菌中得到了融合表达,分子质量约为18 ku,表达量为菌体总蛋白的35%左右。以上结果为下一阶段兔IFN-γ重组蛋白生物学特性及其应用的研究奠定了基础。

Japanese White rabbits IFN-γ gene was amplified by reverse transcription polymerase chain reaction（RT-PCR） from total RNA of the peripheral blood lymphocytes（PBMC） stimulated by ConA.Then the products of RT-PCR were cloned into pGEM-T easy vector.The results indicated that IFN-γ gene of Japanese White rabbits included an opening read frame consisted of 504 bp,which encoded for a polypeptide of 168 amino acids.Compared with other rabbit species in GenBank,including Angora rabbit,Chinese White rabbit,Europen rabbit,and New Zealand rabbit,the homology of nucleotide sequence of IFN-γ gene was 100%;but was 99.8% when compared with Rex rabbit.The host E.coli strain BL21（DE3） transformed with recombinant pET-IFN-γ could express a recombinant protein with molecular weight of 18 ku under the induction of IPTG,which amounted to 35% in the total protein of the induced bacteria by the assaying of gel scanning.This provides a well foundation for further research on rabbit IFN-γ biological properties and applications.