鹅源新城疫病毒M基因的原核表达及其多克隆抗体制备

试验旨在制备原核表达鹅源新城疫病毒(NDV)JS/5/05/Go株M蛋白的多克隆抗体并进行鉴定。以鹅源NDV总RNA为模板,RT-PCR扩增M基因,亚克隆到原核表达载体pET-32a(+)获得重组质粒pET32a-M,转化E.coli BL21(DE3)菌株进行诱导表达,SDS-PAGE电泳检测表达产物;用KCl染色切胶纯化法纯化重组蛋白;采用切胶免疫小鼠的方法制备M蛋白多克隆抗体,抗体效价用间接ELISA检测,特异性用Western blot和间接免疫荧光法鉴定。结果表明,在大肠杆菌中成功表达了分子量约为55 000的重组蛋白,用切胶纯化法获得了纯度较高的重组蛋白;ELISA法检测抗体效价可达1∶102 400,Western blot和间接免疫荧光试验结果表明制备的多克隆抗体能够特异性识别纯化的M蛋白及NDV自身表达的M蛋白。

Prokaryotic expression of M gene of goose-origin Newcastle disease virus and preparation of polyclonal antibodies against M protein

The objective was to express the M protein of goose-origin Newcastle disease virus from its prokaryotic cells and prepare polyclonal antibodies.The M gene was amplified by RT-PCR with a pair of specific primers,and then was subcloned into the prokaryotic expression vector pET-32a(+) to construct the recombinant plasmid pET32a-M.After being transformed into the host strain Escherichia.coli BL21(DE3) and induced by IPTG,the recombinant M protein with relative molecular mass about 55 000 was successfully expressed in E.coli.The target protein was isolated through cutting gel slice containing the right brand which was stained by KCl.The gel slice grinded and dissolved in PBS was then used as antigen to immunize mice for three times to raise polyclonal antibody.Antiserum was analyzed by ELISA,Western blot and indirect immunofluorescence assay(IFA).The results indicated that the M gene was successfully expressed in E.coli BL21(DE3).The ELISA titer of antiserum from vaccinated mice reached 1∶102 400.Western blot analysis and IFA assay showed that the polyclonal antibody could bind to the expressed M protein specifically.