【摘要】 通过反转录-聚合酶链式反应(RT-PCR)扩增了猪瘟病毒(CSFV)的E0、E2和E012基因并进行了克隆与鉴定。构建了真核表达载体pcDNA-E0、pcDNA-E2和pcDNA-E012,通过与鼠白血病病毒(MuLV)假型病毒构建体系的两种质粒pHIT60和pHIT111瞬时共转染人胚肾细胞(293T),48h后收集假型病毒上清液,将假型病毒上清液超速离心后用抗CSFV的多抗进行Western-blot检测。结果证明E012蛋白能够在假型病毒颗粒表面表达,说明E012能够整合到此病毒粒子表面;用其感染多种宿主细胞,48h后检测发现在猪肾细胞(SK6,PK15)和猪睾丸细胞(ST)上,标记基因LacZ能有效表达,证实构建的CSFV假型病毒具有感染性。更多还原

【Abstract】 A transient three-plasmid expression system for the production of pseudotyping virions was used to construct the pseudotype of murine leukemia virus(MuLV)with classical swine fever virus(CSFV)glycoprotein.Co-transfected were pcDNA-E0,pcDNA-E2,pcDNA-E012,pHIT60(including MuLV structural genes,namely gag and pol)and pHIT111(retroviral genome,containing Lac Z as a reporter)into 293T cells.The retroviral supernatants were harvested 48 hours post-transfection,filtered through a 0.45 μm filter.The supernatant was used to analyse the characteristic of the pseudotyping virions by Western-blot and infection test.Western-blot revealed that E012 could be expressed on the virions,indicating the glycoprotein E012 was incorporated onto the retroviral virions.SK6,PK15 and ST infected were Lac Z positive,indicating viral entry,and revealed the pseudtype virions of MuLV-E012 were infectious.The pseudotype system of MuLV particles with CSFV E012 was set up and it can be used to study the entry of CSFV.更多还原