重组鸡α干扰素/白细胞介素-2融合蛋白体外活性评价方法研究

Study on the Activity Estimation Methods of Recombinant Chicken Interferon-α/Interleukin-2 Fusion Protein in vitro

为建立稳定、便捷的重组鸡α干扰素/白细胞介素-2融合蛋白(rChIFN-α-Linker-ChIL-2蛋白,重组融合蛋白)体外活性评价方法,本研究分别采用ChIFN-α和ChIL-2ELISA方法检测重组融合蛋白与抗ChIFN-α单抗和抗ChIL-2单抗发生特异性免疫反应的活性;采用细胞病变抑制法检测重组融合蛋白在DF1细胞上抑制水疱性口炎病毒(VSV)和传染性法氏囊病病毒(IBDV)增殖活性;采用MTS法分别测定重组融合蛋白促鸡外周血T淋巴细胞(PBLC)和脾淋巴细胞增殖活性。结果表明,重组融合蛋白可以与抗ChIFN-α单抗和抗ChIL-2单抗发生特异性免疫反应;重组融合蛋白在DF1细胞上具有明显抗病毒活性,其抗VSV活性高于抗IBDV活性,且均明显高于rChIFN-α蛋白对照;不同浓度的重组融合蛋白均具有明显的促鸡PBLC和脾淋巴细胞增殖活性,且其促增殖活性明显高于rChIFN-α蛋白对照。本研究成功建立了重组融合蛋白体外活性检测评价方法,为进一步探究重组融合蛋白在鸡体内协同作用奠定基础。

In order to establish a stable,simple and rapid detection method to estimate the activities of the recombinant chicken interferon-α/interleukin-2fusion protein（rChIFN-α-Linker-ChIL-2,recombinant fusion protein）in vitro,the activities of rChIFN-α-Linker-ChIL-2were estimated by detecting its specific immune response to monoclonal antibody（MAb）against ChIFN-αand ChIL-2by ELISA assay.The antiviral activities of rChIFN-α-Linker-ChIL-2protein were tested by inhibiting the 50% appearance of cytopathic effect（CPE）of vesicular stomatitis virus（VSV）and infectious bursal disease virus（IBDV）on the passage cell lines DF1.The promoting proliferation activities of lymphocytes in the chicken peripheral blood and spleen of recombinant fusion protein were tested by MTS method.The results showed that rChIFN-α-Linker-ChIL-2protein had the ability of specific immune response to anti-ChIFN-α MAb and anti-ChIL-2 MAb,respectively.The antiviral activity of rChIFN-α-Linker-ChIL-2protein inhibiting the reproduction of VSV on DF1 cell line was higher than IBDV,and both of the antiviral activities of recombinant fusion protein against VSV and IBDV were much higher than the recombinant ChIFN-αprotein（rChIFN-α）control.The recombinant fusion protein had apparent promoting proliferation activity of lymphocytes in the chicken peripheral blood and spleen,which were much higher than that of the rChIFN-αcontrol.The study suggested that the activities detection and estimation methods of the recombinant fusion protein in vitro were successfully established,which laid the foundation for the further study of the synergy activity of recombinant fusion protein in vivo.