鸡粒细胞巨噬细胞集落刺激因子-猪干扰素α1融合基因在大肠埃希菌中的可溶性表达及其生物学活性研究

Soluble expression and activity analysis of chicken granulocyte macrophage-colony stimulating factor-porcine interferon alpha 1 fusion gene in Escherichia coli

为可溶性表达重组鸡粒细胞巨噬细胞集落刺激因子(ChGM-CSF)与猪干扰素α1(PoIFNα1)融合蛋白,并研究其生物学活性,本研究分别提取鸡、猪肝脏细胞总RNA,采用RT-PCR方法扩增ChGM-CSF和PoIFNα1基因,经linker连接上述两种基因后将其克隆于pET-32a原核表达载体,转化E.coliBL21(DE3)菌株进行诱导表达,SDS-PAGE和western blot检测融合蛋白表达产物。在ST细胞/VSV病毒测定系统以细胞病变抑制法滴定该融合蛋白的抗病毒活性;同时用MTT法检测其对鸡淋巴细胞增殖活性的促进作用。结果显示,PCR扩增并融合后的ChGM-CSF和PoIFNα1融合基因约为1000bp,构建重组表达载体后,诱导表达的rChGM-CSF-PoIFNα1融合蛋白分子量约55ku,主要存在于破碎菌体的上清中,表达量较高。Westernblot检测结果显示,该融合蛋白分别能够与ChGM-CSF多抗和PoIFNα单克隆抗体特异性结合,其抗病毒比活性约为1.1×10^6IU/mg,并且具有促进鸡淋巴细胞增殖的活性。本研究为rChGM-CSF-PoIFNα1重组融合蛋白的研制及其相关活性的测定提供实验依据。

To investigate the biological activities of chicken granulocyte-macrophage colony-stimulating factor (ChGM-CSF) and porcine interferon alpha 1 (PoIFNα1), the recombinant plasmid with both two genes above was expressed in vivo. In this study, these two genes were amplified by RT-PCR from the total RNAs of chicken and porcine liver cells respectively before connecting with each other by a linker. And the resulting fusion gene was inserted into pET-32a prokaryotic expression vector and its recombinant plasmid was transformed into E.coli BL21 (DE3) strain. The expression products were subjected to SDS-PAGE and western blot, and their antiviral activity was determined using the ST cell/VSV assay system with the method to the cytopathic effect (CPE) reduction assay. Additionally, the MTT assay was performed to detect the activity of the recombinant protein in term of promoting the proliferation of chicken lymphocytes. The results showed that the lengths of the fusion gene was about 1,000bp with a linker involved. The fusion gene was clone and expressed sucessfully in E.coli. The molecular weight of recombinant protein was 55ku. The expressed proteins were mainly found to be water soluble with a good expression level. The protein could bind specifically to the monoclonal antibodies against either rChGM-CSF or rPoIFNα, and its antiviral specific activity was about 1.1×10^6 IU/mg. And it has an effect on promoting lymphocyte proliferation in chicken blood. This current study will provide some experimental experiences for the expression and activity analysis of the rChGM-CSF-PoIFNα1 recombinant fusion protein.