摘要
通过RT-PCR从经ConA刺激诱导的奶牛脾脏淋巴细胞总RNA中扩增出牛γ干扰素(BoIFN-γ)cDNA,克隆到真核载体pVAX1中,测序结果显示pVAX1中的插入序列BoIFN-γ基因与已报道序列一致。用重组质粒pVAX1-BoIFN-γ转染COS-7细胞并进行间接免疫荧光试验鉴定,结果显示BoIFN-γ在COS-7细胞中得到成功表达。将BoIFN-γ基因克隆到原核表达质粒pET-30a(+)、pGEX-6p-1后,分别转化重组表达菌BL21(DE3)、BL21后,通过对表达条件的优化,SDS-PAGE分析表明两种重组蛋白均可实现可溶性表达,大小分别为23 kDa和43 kDa。将含信号肽BoIFN-γ基因克隆到转座载体pFastBacTM1中并转化DH10Bac,通过位点特异性转座将BoIFN-γ基因整合到穿梭载体Bacmid中并通过脂质体转染Sf9昆虫细胞,产生重组杆状病毒rBac-BoIFN-γ,重组病毒传代扩增感染Sf9细胞后,通过间接免疫荧光试验证实BoIFN-γ在杆状病毒系统中获得表达。使用MDBK/VSV细胞系统测定rHis-BoIFN-γ、rGST-BoIFN-γ以及rBac-BoIFN-γ的抗病毒活性,其效价分别达到8.389×107 U/mg、6.554×105 U/mg、4.096×104 U/mL,3种具有较高的抗病毒活性重组牛γ干扰素的获得为其开发应用提供了重要的生物材料。同时使用单克隆抗体5G4和3E6,建立了BoIFN-γ的双抗夹心ELISA检测方法并绘制了标准曲线,可定量分析BoIFN-γ的抗病毒活性,为其临床应用及相关研究提供了重要的方法。
Bovine interferon-γ（BoIFN-γ） gene was amplified by reverse transcription polymerase chain reaction（RT-PCR） from total RNA of bovine spleen lymphocytes stimulated with ConA.The products of RT-PCR were cloned into pVAX1 vector,positive recombinant clone was identified by restriction enzyme digestion and sequencing.The recombinant plasmid pVAX1-BoIFN-γ was transfected into COS-7 cells mediated by lipofectine,indirect immunofluorescent assay analysis confirmed that rBoIFN-γ was expressed in COS-7 cells.BoIFN-γ gene（without signal peptide） was cloned into pET-30a（＋） and pGEX-6p-1 vector,and transformed into the Escherichia coli cells.After optimizing the induction condition,SDS-PAGE analysis showed that the expression products were all found in soluble form and had a molecular weight of 23 kDa and 43 kDa respectively.BoIFN-γ precursor gene（with signal peptide） was cloned into transfer vector pFastBacTM1,and transformated into DH10Bac E.coli cells.By site-specific transposition,BoIFN-γ gene was integrated into shuttle vector Bacmid,and transfected into the Sf9 insect cells mediated by lipofectine to produce recombinant baculovirus.Indirect immunofluorescent assay analysis confirmed that rBac-BoIFN-γ was expressed successfully in Baculovirus vector system.The antiviral activities of rHis-BoIFN-γ,rGST-BoIFN-γ and rBac-BoIFN-γ were up to 8.389×107 U/mg,6.554×105 U/mg and 4.096×104 U/mL respectively,which were analyzed in MDBK/VSV system.A sandwich ELISA was established using monoclonal antibodies 3E6 and 5G4,which can detect BoIFN-γ in quantity and provide a useful method for the clinical practice and research of BoIFN-γ.
出处
《生物工程学报》
CAS
CSCD
北大核心
2011年第2期269-276,共8页
Chinese Journal of Biotechnology
基金
国家重点基础研究发展计划(973计划)(No.2006CB504404)
国家科技重大专项(No.2008ZX10003-010)
公益性行业科研专项(No.200903027)资助~~
