猪γ-干扰素全基因克隆及原核表达

摘要

To amplify pig interferon-y(pIFN-y) cDNA from ConA-activated peripheral-blood lymphocyte by RT-PCR, then was cloned into pMD18T and sequenced, and recombined with pET32a( + ) vector, the target protein HisX6 IFN-γ was induced by IPTG;the sequence analysis showed that the nucleotide sequences of the IFN-γ in the study shared 98. 6% to 100. 0% homology with that of pig published in GenBank, and the identity of amino acid was 96. 4% to 99. 4%. This recombinant protein , a molecular weight of about 27 ku and mainly presenting in inclusion bodies, was approved to have immunological activity by Western blotting analysis and SDS-PAGE. It laid a foundation for further research of IFN-γ.%利用RT-PCR技术,从ConA活化的猪外周血淋巴细胞中扩增出γ-干扰素(interferon-γ,IFN-γ) cDNA,克隆到pMD18-T载体中进行测序后,与原核表达载体pET32a(+)重组,表达含His×6的IFN-γ重组蛋白；测序结果与GenBank中已发表的序列进行比对,核苷酸同源性在98.6％～100.0％之间,氨基酸同源性在96.4％～99.4％；经SDS-PAGE和Western blotting分析结果表明,该重组蛋白分子质量约为27 ku,主要存在于包涵体中,且具有良好的免疫学活性,为进行γ-干扰素深入的研究奠定了基础.