新城疫强毒株F蛋白裂解位点串联基因的构建及其原核表达

Construction of Prokaryotic Expression Vector for F Protein Schizolysis Site Gene Cascade of Virulent Newcastle Disease Virus Strain

目的构建鸡新城疫(ND)强毒株多裂解位点串联基因表达载体,为建立NDV强弱毒株的ELISA鉴别方法奠定基础。方法人工合成一段包含4种不同NDV强毒株裂解位点的基因Fe(F prote in multitude epi-position),其中各裂解位点之间用Linker(GGGGS)使其串联,然后进行2倍和4倍拷贝,并定向克隆至原核表达质粒pET-28a(+)中,构建重组表达质粒pET-28a(+)/2Fe和pET-28a(+)/4Fe,进行表达和Western blot鉴定。结果构建的原核表达重组质粒pET-28a(+)/2Fe和pET-28 a(+)/4Fe测序正确。Western blot检测结果表明,2Fe、4Fe蛋白均与新城疫强毒株F48E9阳性血清呈阳性反应,而与新城疫弱毒株V4阳性血清呈阴性反应。结论已成功构建NDV强毒株F蛋白裂解位点串联基因表达载体,为建立NDV强弱毒株的ELISA鉴别方法提供理论依据。

Objective To construct the prokaryotic expression vector for F protein schizolysis site gene cascade of virulent Newcastle disease virus（NDV） strain and lay a foundation of differentiation of virulent and low virulent NDV strains by ELISA. Methotis Synthesize a gene fragment Fe （ F protein multitude epi-position） containing 4 different schizolysis sites of virulent NDV strains, of which the sites were linked through a linker GGGGS. Copy Fe gene for 2 and 4 times,then clone the obtained 2Fe and 4Fe genes directly into pmkaryotic expression vector pET-28a（ ＋ ） respectively. Transform the constructed recombinant plasmids pET-28a（ ＋ ）/2Fe and pET-28a（ ＋ ）/4Fe to E. coil BL21 （DE3） respectively and identify the expressed products by Western blot. Results Sequencing results proved that recombinant plasmids pET-28a（ ＋ ）/2Fe and pET-28a（ ＋ ）/4Fe were correctly constructed. Western blot shooed specific reaction of expressed 2Fe and 4Fe proteins with F48E9 positive sera but no reaction with V4 positive sera. Conclusion A prokaryotic expression vector for F pretein schizolysis site gene cascade of virulent NDV strain was successfully constructad,which provided a theoretical basis for differentiation of virulent and low virtdent NDV strains by ELISA.