重组鸡γ_干扰素在昆虫细胞中的高效表达

将鸡γ-干扰素(ChIFN-γ)基因克隆到载体pFASTBAC1中,构建转移载体pFASTBAC1-ChIFN-γ,然后转化DH10Bac感受态大肠杆菌,通过位点特异性转座,将ChIFN-γ基因整合到Bacmid穿梭载体中,构建表达质粒Bacmid-ChIFN-γ;通过脂质体将表达质粒转染Sf9昆虫细胞,用间接免疫荧光试验鉴定重组鸡γ-干扰素(rChIFN-γ)的表达;通过水泡性口炎病毒感染鸡胚成纤维细胞(CEF)的细胞病变抑制试验,检测rChIFN-γ的活性。研究结果表明,感染重组杆状病毒的Sf9细胞能高效表达rChIFN-γ,且当每个细胞的感染量为1个病毒时,细胞在感染96h后,rChIFN-γ基因表达产物的活性最高,达到106~107.2U/mL。以rChIFN-γ进行对新城疫病毒(NDV)F48E8株、禽流感H5N1病毒(AIV-H5)和马立克氏病病毒(MDV)GA株的抗病毒活性试验,发现rChIFN-γ对AIV-H5和MDV GA株病毒有明显的抑制其致细胞病变作用,但对NDVF48E8株病毒在体外不能抑制其致细胞病变作用,仅能在病毒滴度上表现抑制效果。

High level expression of recombinant chicken interferon-gamma in insect cells

The recombinant transfer vector pFastBacl-ChIFN-y was constructed by plasmid pcDNA-ChIFN-gamma digested with EcoR I and Not I enzymes and cloned into pFastbacl. Then the transfer vector was transformed into E. coli competent cells DH10Bac which contained the bacmid with amini-attTn7 target site and the helper plasmid. The recombinant bacmid-ChIFN-gamma was generated by transposing themini-Tn7 element located in pFastBacl-ChIFN-gamma to themini-attTn7 attachment site on the Bacmid. Subsequently the recombinant Bacmid-ChIFN-gamma was transfected into the Sf9 insect cells mediated by lipofectin to produce recombinant baculovirus and express recombinant ChIFN-gamma (rChIFN-gamma) products. The result showed that the rChIFN-gamma was successfully expressed in Sf9 cells infected with the recombinant virus by indirect immunofluorescence assay (IFA) at 5 days post-transfection. The biological activity of rChIFN-gamma was identified by its inhibition to Vesicular stomatitis virus-induced cytotoxicity of chicken embryonic fibroblasts (CEF) in vitro. The results showed that the most efficient expression of rChIFN-gamma could be obtained at 96h post-infection with multiplicity of infection (MOI) equal to 1. It is interesting that the viruses such as Avian influenza virus H5N1 or Marek's disease virus (GA strain) could not grow in CEF pre-treated with rChIFN-gamma. Cell pathogenic efficient (CPE) in the CEF infected with H5N1 and GA strain is apparently inhibited by the rChIFN-gamma. However only difference between the HA titres of the supernatant of the pre-treated cells is observed without any obvious inhibition effect in CEF infected with Newcastle disease virus (F48E8 strain).