摘要
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27ku,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。
To amplify pig interferon-γ（pIFN -γ） cDNA from ConA activated peripheral-blood lymphocyte by RT PCR, then was cloned into pMD18-T and sequenced, and recombined with pET32a（＋） vector, the target protein His× 6 IFN-γ was in duced 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 pro- rein, 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.
出处
《中国畜牧兽医》
CAS
北大核心
2012年第10期91-94,共4页
China Animal Husbandry & Veterinary Medicine
关键词
基因克隆
原核表达
IFN-Γ基因
猪
gene cloning
prokaryotic expression
interferon-γ gene
pig
