表达H5N1亚型禽流感病毒HA蛋白的重组鼠白血病病毒的特性

Characterization of Murine Leukemia Virus Recombinants that Express H5N1 Subtype Avian Influenza Virus Hemagglutinin Glycoproteins

通过反转录 聚合酶链式反应 (RT PCR)扩增了H5N1亚型鹅源禽流感病毒 (AIV)完整的血凝素 (HA)基因并进行了克隆与鉴定。序列测定结果已经登陆GenBank ,登陆号为AY6 394 0 5。序列分析表明所扩增的HA基因开放性阅读框架 (ORF)由170 7个核苷酸组成 ,共编码 5 6 8个氨基酸 ,裂解位点的氨基酸组成为RKKR↓GLF ,含连续的碱性氨基酸 ,具有高致病性AIVHA基因裂解位点的特征。构建了含HA基因的真核表达载体pcDNA HA ,通过与鼠白血病病毒 (MuLV)假病毒构建体系的两种质粒pHIT6 0和pHIT111共转染人胚肾细胞 2 93T ,4 8h后收集假病毒上清 ,超离后通过Western blot证明HA蛋白能够在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 2 93T、COS 7和NIH3T3三种不同的靶细胞 ,证实所构建的假病毒粒子具有感染性和泛嗜性。本研究成功构建了具有感染性的MuLV HA假病毒体系 ,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。

One highly pathogenic strain of avian influenza virus (AIV) was isolated from goose in China recently, designated as F 3 In order to study the viral entry mechanisms, the hemagglutinin(HA) gene of H5N1 subtype AIV isolate was amplified by RT PCR, and then cloned into pGEM D○R T vector and sequenced. The sequencing result has logging in GenBank, the accession number was AY639405 The HA gene of F 3 had a complete open reading frame (ORF) and composed of 1707 nucleotides, coding for 568 amino acids. The deduced amino acid sequence at the cleavage site of the HA protein was RKKR↓GLF, matched to the characteristic of virulent avian influenza strain. The HA gene were subcloned into pcDNA3, so the plasmid pcDNA HA can express the HA glycoprotein. Co transfected pcDNA HA, pHIT60(include Murine Leukemia Virus structural genes,namely gag and pol) and pHIT111(retroviral vector genome,containing LacZ as a reporter) into 293T cells. The retroviral supernatant were harvested 48 hours post transfection, filtered through 0 45μmol/L 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 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 lacZ positive, indicating viral entry, and revealed the pseudotype virions of MuLV HA were infectious. So the pseudotype system of MuLV particles with AIV Hemagglutinin proteins were setted up and it can be used to study the entry of avian influenza virus isolated from goose in China.