摘要
本文应用(RT-PCR)技术,从猪瘟弱毒疫苗和Poly:IC共刺激7 h的PK15细胞的cDNA中扩增出编码Mx1蛋白的全长基因,并通过引物设计填补了PK(15)细胞系Mx1 cDNA序列中3'端11bp的缺失,成功获得Mx1完整基因的克隆。构建了重组表达质粒pRetroQ-sMx1,转染HEK293T细胞并表达Mx1蛋白,利用微量细胞病变抑制法测定其抗病毒活性。双酶切鉴定和核酸序列测定证实pRetroQ-sMx1真核表达质粒构建成功,转染HEK 293T细胞后,能够检测到绿色荧光,Western blot证实为目的蛋白。微量细胞病变抑制法测定重组蛋白具有一定的抗水疱性口炎病毒及猪繁殖与呼吸综合征病毒的活性。重组Mx1具有一定抗病毒生物功能,为进一步研究重组Mx1蛋白的活性以及Mx1抗病毒药物奠定了基础。
The present study was to clone Myxovirus resistance protein Mx1 for eukaryotic expression and detect its antiviral activity. The RNA coding for Mx1 was extracted from PK15 cells that were stimulated for 7 hours with swine fever vaccine and Poly real：IC. Subsequently, full-length Mx1 gene was amplified in RT-PCR using primers designed to rescue 11 bp missing in PK 15 cell line. The recombinant expression plasmid pRetroQ-sMx1 was constructed and used to transfect HEK293T cells for expression. Success of Mx1 expression in HEK293T cells was confirmed through double enzyme identification, determination of nucleic acid sequence, detection of green fluorescence and Western blot. Cytopathic effect inhibition assay was used to measure antiviral activity of expressed Mx1.
出处
《中国动物传染病学报》
CAS
2014年第4期35-39,共5页
Chinese Journal of Animal Infectious Diseases
基金
山东省科技攻关项目(2009GG10009011)
