Hartman J R, Nayak D P, Fareed G C
Proc Natl Acad Sci U S A. 1982 Jan;79(2):233-7. doi: 10.1073/pnas.79.2.233.
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.
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，在猴肾细胞中克隆并表达了该基因。SV40基因组包含完整的早期基因区域、复制起点和晚期前导序列。HA基因（长1775个碱基对）最初通过dG·dC加尾技术，利用从病毒RNA制备的cDNA插入大肠杆菌的多拷贝质粒pBR322中。克隆的基因经核酸酶Bal 31处理，去除dG·dC尾和一些非翻译序列，然后在添加BamHI限制性内切酶接头后重新克隆到大肠杆菌中。通过将重新克隆的HA DNA插入SV40的晚期基因区域，构建了许多SV40和HA重组体（SV--HA）。当SV40早期缺失突变体的辅助功能在猴细胞的裂解感染中对SV--HA重组体进行互补时，使用异种抗流感兔抗体或抗HA单克隆抗体通过免疫荧光和体内标记蛋白的免疫沉淀检测到表达了流感HA。此外，SV--HA重组体表达的WSN HA也进行了糖基化，并且在猴细胞中与WSN病毒未切割的HA具有相同的分子量（约70,000）。
