重组扬州鹅IFN-α的表达与活性研究

Expression and activity analysis of recombinant Yangzhou goose IFN-α

为获得具有抗病毒活性的鹅干扰素,本实验参考GenBank中鹅IFN-α基因序列设计引物,采用RT-PCR方法扩增扬州鹅IFN-α的全基因。将去除信号肽的IFN-α克隆至pET32a(+)以构建pET-mGoIFN-α,重组菌经IPTG诱导表达重组蛋白,纯化后的蛋白(mGoIFN-α)免疫BALB/c小鼠,利用ELISA方法测定小鼠血清抗体效价,westernblot检测抗体特异性。同时,将IFN-α全长基因克隆至pcDNA3.1(-)以构建pcDNA-GoIFN-α,间接免疫荧光试验检测rGoIFN-α在鹅胚成纤维细胞(GEF)中的表达,细胞病变抑制法检测其抗水泡性口炎病毒(VSV)活性。结果显示:本研究扩增得到576bp的扬州鹅IFN-α全基因与486bp的成熟基因片段;原核表达的mGoIFN-α为36.3ku,免疫小鼠后可产生具有良好特异性与抗原活性的多克隆抗体,效价为1:16000;pcDNA-GoIFN-α可在GEF中表达,并具有较高的抗VSV活性。本研究为制备安全、高效的重组鹅干扰素,为研制鹅病毒性疾病的新型生物制剂奠定了基础。

In order to obtain goose interferon with antiviral activity,the primers were designed based on the goose IFN-αgene sequence published in GenBank amplified the whole gene of Yangzhou goose IFN-αwas obtained by RT-PCR amplification.The IFN-αgene with exclusion of signal peptide sequence was cloned into pET32a(+)vectors,and the recombinant plasmid of pET-mGoIFN-αwas constructed.The recombinant protein was induced by IPTG and purified for BALB/c immunizing.The antibody titers of immune serum was determined by ELISA.The specificity of immune serum was tested by western blot.At the same time,the whole gene of IFN-αwas cloned into pcDNA3.1(-)to construct the recombinant plasmid of pcDNA-GoIFN-α.The expression of rGoIFN-αin goose embryo fibroblasts(GEF)was analyzed by indirect immunofluorescence assay,and the cytopathic inhibition assay was performed for the anti-VSV activity detection of rGoINF-αprotein.The results showed that both the whole and mature gene of IFN-αwere successfully amplified,with the size of 576 bp and 486 bp respectively.The size of prokaryotic expressed mGoIFN-αwas 36.3 ku,The immune serum in mice included specific antibodies against m GoINF-αprotein and had good reactivity with mGoINF-αprotein.The antibody titer was 1:16000.The full-length gene of GoIFN-αcan be expressed in cells,and the resulting protein can effectively inhibite the VSV infection.The study laid the foundation for the preparation of safe and efficient GoIFN-αand the development of new biological agents for the prevention and treatment of goose viral diseases.