关键词: face=Verdana>鸡α干扰素, 鸡白细胞介素2, 嵌合基因, 融合表达, 活性

Abstract: 【Objective】 The aim of this study was to explore a high efficient chicken gene engineering antiviral agent to prevent and control the chicken viral disease. 【Method】 The recombinant chimeric gene of ChIFN-α-linker-ChIL-2 constructed by chicken interferon α ( ChIFN-α) gene linked chicken interleukin-2 (ChIL-2) gene via a glycine-rich linker by SOE-PCR (splicing by overlap extension-PCR) method was cloned into pGEM-T Easy vector and subsequently sub-cloned into prokaryotic expression vector pQE30. The recombinant ChIFN-α-linker-ChIL-2 (rChIFN-α-linker-ChIL-2) protein was expressed in E.coli JM109 and purified with the innovated recombinant fusion protein purification method and denatured by 8 M urea, refolded by a self-innovative renaturation buffer and dialyzed by PBS buffer, etc. The antiviral bioactivities of rChIFN-α-linker-ChIL-2 protein were tested by inhibiting the 50 percent appearance of cytopathic effect (CPE) of vesicular stomatitis virus (VSV) and infectious bursal disease virus (IBDV) on chicken embryo fibroblast (CEF) cell lines. The ChIL-2 bioactivities of rChIFN-α-linker-ChIL-2 protein were estimated by the method of chicken IL-2 ELISA assay for detection of the specific immunoactivity of ChIL-2 protein. The antivirus bioactivity of rChIFN-α-linker-ChIL-2 protein was evaluated by inhibiting the reproduction of NDV and AIV (H9N2) in chicken embryos, respectively. The bioactivity of rChIFN-α-linker-ChIL-2 protein in chicken was estimated by detecting the NDV hemagglutination inhibition (HI) antibody titer induced by NDV oil-adjuvant inactivated vaccine and attenuated live vaccine, respectively. 【Result】 The chimeric gene of ChIFN-α-linker-ChIL-2 was successfully constructed and cloned into pGEM-T Easy vector, respectively. The rChIFN-α-linker-ChIL-2 protein was abundantly fusion expressed in E.coli and successfully purified with a molecular mass of about 35.9 kD and purity more than 96% on SDS-PAGE, which indicated that the correct rChIFN-α-linker-ChIL-2 fusion protein had been obtained. The antiviral activity units of rChIFN-α-linker-ChIL-2 protein inhibiting the reproduction of VSV and IBDV on CEF cell line were 1.14×108IU?mg-1 and 1.59×106 IU?mg-1 ,respectively, which were much higher than that of the recombinant ChIFN-α protein (rChIFN-α) of control (4.09×106 IU?mg-1, 2.78×105 IU?mg-1). The rChIFN-α-linker-ChIL-2 protein showed the ability of the specific immune response to monoclonal antibody (MAb) of ChIL-2 by ELISA assay, which was similar to the bioactivity of recombinant ChIL-2 protein (rChIL-2) of control. The rChIFN-α-linker-ChIL-2 protein with 200 international unit of IFN-α could obviously decrease the death rate and haemorrhage degree as well as prolong the living time of chicken embryos inoculated by NDV and AIV H9N2, whereas the excessive dose of rChIFN-α-linker-ChIL-2 protein displayed the lower ability of decreasing the death rate and haemorrhage degree of chicken embryos. The rChIFN-α-linker-ChIL-2 protein with appropriate dose exhibited the prominent abilities of antivirus and immunopotentiation in chicken.【Conclusion】 The results of study indicated that the rChIFN-α-linker-ChIL-2 protein had the duplex bioactivity of ChIFN-α protein and ChIL-2 protein on cells, in chicken embryos and chicken, thus laid a foundation for further widely use of rChIFN-α-linker-ChIL-2 protein acting as a main ingredient of chicken gene engineering antiviral agent to prevent and control the chicken viral diseases.

Key words: chicken interferon alpha, chicken interleukin-2, chimeric gene, fusion expressed, bioactivity