【摘要】 利用RT-PCR技术从有丝分裂原刀豆素(ConA)诱导的长白猪外周血淋巴细胞总RNA中扩增出猪γ干扰素的成熟肽基因(mPoIFN-γ),并将其亚克隆到原核表达载体pET-28a中,构建重组表达载体pET-mPoIFN-γ。经PCR、酶切及测序鉴定后,转化到大肠杆菌BL21(DE3)进行IPTG诱导表达,经SDS-PAGE和Western-blot检测,表明猪γ干扰素获得了高效表达,并具有免疫活性,表达产物约为20.5 kDa,主要以包涵体形式存在,产物经纯化、复性得到具有生物学活性的蛋白。利用细胞病变抑制法对其在H-PRRSV/Marc-145系统上进行抗病毒活性测定,结果表明其抗H-PRRSV活性为7.68×103 U/mg。为进一步研究猪γ干扰素功能奠定了基础和理论依据。更多还原

【Abstract】 For constructing recombinant expression vector pET-mPoIFN-γ,the mature peptide gene for porcine interferon-γ(mPoIFN-γ) was amplified by RT-PCR depending on the template of total RNA which was isolated from the peripheral blood lymphocyte of ConA-stimulated Landrace swine,and then subcloned into pET-28a vector.After detection by PCR,restriction enzyme digestion and sequencing,the recombinant plasmid was transformed into Escherichia coli BL21(DE3) and inducted by IPTG.The SDS-PAGE analysis and Western blot results showed that the porcine interferon-γ was highly expressed with immune activity and the expressed product was about 20.5kDa,was existed mainly in inclusion body.The recombinant product was purified and renaturalized from inclusion body to obtain the protein with biological activity.Antiviral activity assay for PoIFN-γ was performed and evaluated by standard procedures in H-PRRSV/Marc-145(virus/cell) test system.The results showed that the titer of PoIFN-γ against H-PRRSV was 7.68×103 U/mg.The research laid a foundation for the function investigation of PoIFN-γ.更多还原