猪γ干扰素的克隆与序列分析及腺病毒表达载体的构建

Cloning and Sequence Analysis of Porcine Interferon Gamma Gene and Construction of Recombinant Adenovirus Vector Containing IFN-γ

提取猪脾脏淋巴细胞中总RNA,应用RT-PCR技术扩增出γ-干扰素基因,将其克隆到pGEM-T载体上,并进行测序。结果表明,克隆获得的γ-干扰素基因全长为517个碱基,ORF为501个碱基,编码166个氨基酸。与GenBank上已发表的猪γ-干扰素基因的核苷酸同源性均为100%,并且在氨基酸水平上也完全一致。将目的基因插入腺病毒穿梭质粒pAd-Shuttle-CMV载体中,构建出真核表达载体pAd-Shuttle-CMV-IFNγ-,在BJ5183细菌中同源重组,构建出腺病毒表达载体pAdenoγ-,为进一步研究γ-干扰素在腺病毒中的表达、检测其生物学活性奠定基础。

The total RNA was isolated from spleen lymphocytes from swine, then the IFN-γ gene was amplified via RT-PCR. The gene segment of IFN-γ was cloned into the vector pGEM-T. From the sequence, it was found that the IFN-γ gene was consisted of 517 nucleotides. The ORF was consisted of 501 nucleotides, encoding 166 amino acids. The homology of the IFN-γ gene was 100% at nucleotide level and amino level compared with other porcine IFN-γ sequence published on GenBank. The objective gene was cloned into pAd-Shuttle-CMV vector and constructed the eukaryotic expression plasmid pAd-Shuttle-CMV-IFN-γ. The positive recombinant pAd-Shuttle-CMV-IFN-7 was linearized and transformed into Ad-BJ5183 competent cells which had already been transformed with adenovirus skeletal vector pAdeasy-1. The Adenovirus expression plasmid was confirmed by Pac Ⅰ digestion. These results indicated that the recombinant adenovirus plasmid was successfully constructed and established basic work for further study.