Human influenza virus hemagglutinin is expressed in monkey cells using simian virus 40 vectors.
Human influenza virus hemagglutinin is expressed in monkey cells using simian virus 40 vectors.
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使用猿猴病毒 40 载体在猴细胞中表达人流感病毒血凝素。
DOI：
10.1073/pnas.79.2.233
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发表时间：
1982
影响因子：
11.1
通讯作者：
Fareed,GC
中科院分区：
文献类型：
--
作者：
Hartman,JR;Nayak,DP;Fareed,GC
我们通过将人流感病毒(A/WSN/33)的血凝素(HA)基因与缺失的猿猴病毒40(SV40)基因组连接，克隆并在猴肾细胞中表达，该基因组包含完整的早期基因区、复制起点和晚期前导序列。HA基因(1775个碱基对)最初是由DG插入的。采用DC拖尾技术，以病毒RNA为模板，构建了多拷贝表达载体pBR322。用Bal 31核酸酶对克隆的基因进行进一步修饰，去除DG。DC尾和一些未翻译的序列，并在添加BamHI限制性内切酶连接子后在大肠杆菌中重新克隆。将重克隆的HA DNA插入到SV40的晚期基因区，构建了SV40和HA重组体(SV-HA)。在SV40早期缺失突变体的辅助作用下，SV-HA重组体在猴细胞裂解感染中表达流感HA，免疫荧光和体内标记蛋白的免疫沉淀检测表明，无论是异源抗流感兔抗体还是抗HA单抗，都能表达流感HA。此外，SV-HA重组体表达的WSN HA也是糖基化的，与WSN病毒在猴细胞中未裂解的HA具有相同的分子量(约70,000)。
We have cloned and expressed the hemagglutinin (HA) gene of a human influenza virus (A/WSN/33) in monkey kidney cells by linking it to deleted simian virus 40 (SV40) genomes that contain the entire early gene region, the origin of replication, and late leader sequences. The HA gene (1775 base pairs long) was originally inserted by the dG . dC tailing technique into the multicopy plasmid of Escherichia coli, pBR322, using cDNA made from viral RNA. The cloned gene was further modified by treatment with nuclease Bal 31 to remove the dG . dC tails and some of the untranslated sequences and recloned in E. coli after addition of BamHI restriction endonuclease linkers. A number of SV40 and HA recombinants (SV--HA) were constructed by inserting recloned HA DNA into the late gene region of SV40. The SV--HA recombinants, when complemented in a lytic infection of monkey cells by the helper function of SV40 early deletion mutants expressed influenza HA as detected by immunofluorescence and immunoprecipitation of in vivo-labeled proteins using either heterogeneous anti-influenza rabbit antibodies or monoclonal antibodies against HA. Furthermore, the WSN HA expressed by the SV--HA recombinants was also glycosylated and possessed the same molecular weight (approximately 70,000) as the uncleaved HA of WSN virus in monkey cells.