本研究通过一个瞬时转染系统将H5N1亚型鹅源禽流感病毒囊膜表面的血凝素(HA)糖蛋白整合到鼠白血病病毒(MuLV)颗粒表面并进行了感染性测定。将包含HA基因的真核表达质粒pcDNA-HA与MuLV假病毒构建体系的两种质粒pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHIT111(为MuLV的基因组,还包括一个报告基因LacZ)瞬时共转染转化了SV40大T抗原的人胚肾细胞293T,48小时后收集假病毒上清进行了一系列鉴定。将假病毒上清超速离心后用抗H5亚型禽流感病毒的多抗通过Western-blot证实HA 蛋白能够在此假病毒颗粒表面表达,表明HA能够整合到此病毒粒子表面。通过感染293T、COS 7和NIH3T3 三种不同的靶细胞,均能检测到LacZ基因的表达,证实所构建的假病毒粒子具有感染性。本研究成功构建了具有感染性的MuLV-HA假病毒,为研究鹅源禽流感病毒侵入细胞的机理及其组织嗜性的变异提供一种新方法。

A transient three-plasmid expression system for the production of pseudotyping virions were used to construct the pseudotype of murine leukemia virus(MuLV) with avian influenza virus hemagglutinin(HA) glycoproteins. Co-transfected pcDNA-HA, pHIT60(include 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 0.45μM filter. The supernatant were used to analysis the characteristic of the pseudotyping virions by Western blotting and infection test. Western blotting revealed the HA glycoproteins can be expressed on the virions, indicated the HA glycoproteins were incorporated onto the retroviral virions. Infection test were performed on 293T、NIH3T3 and COS-7, all the three kinds of cells infected were Lac Z positive, indicating viral entry, and revealed the pseudotype virions of MuLV-HA were infectious. The pseudotype system of MuLV particles with AIV ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">Hemagglutinin proteins were set up and it can be used to study the ent ry of avian influenza virus isolated f rom goose in China.ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">Hemagglutinin proteins were set up and it can be used to study the ent ry of avian influenza virus isolated f rom goose in China.