H5亚型AIV的HA1基因与鸡IL-18成熟蛋白基因的杆状病毒共表达载体的构建

参考GenBank上发表的H5亚型禽流感(AIV)血凝素(HA)基因序列及鸡白细胞介素18成熟蛋白(mChIL-18)的cDNA基因序列,分别设计了HA1和mChIL-18的cDNA的2对引物。然后以pMD18-T-HA质粒和pGEX-mChIL-18质粒为模板,扩增出了H5亚型AIV的HA1基因和mChIL-18的cDNA片段。再以杆状病毒pFastBacTM dual为载体,将H5亚型AIV HA中的HA1基因和mChIL-18基因分别插入到双表达载体pFastBacTM dual的p10启动子(PP10)和多角体蛋白启动子(PPH)的下游,构建携带HA1基因和mChIL-18基因的重组pFastBacTM dualH-A1-mChIL-18真核表达质粒。最后将构建的真核表达质粒转座入大肠杆菌DH10Bac,为下一步进行昆虫细胞的转染奠定了基础。这个重组载体的构建为考察mChIL-18作为免疫增强剂的作用及下一步AIV疫苗的研究奠定了坚实的基础。

Construction of recombinant baculovirus vetor coexpressing HA1 gene from subtype H5 of avian influenza virus and mature chicken interleukin-18 gene

The hemagglutinin(HA) gene of Avian Influenza Virus(AIV) subtype H5 and mature chicken interleukin-18(mChIL-18) gene were amplified by designing two pairs of primers according to the sequence published on GenBank.Recombinant baculovirus coexpressing HA1 gene from AIV subtype H5 plus chicken interleukin-18 gene were constructed by inserting cDNA copy of HA gene and cDNA copy of mChIL-18 gene into the baculovirus expression plasmid pFast BacTMDual,furthermore cDNAs respectively inserted into the downstreams of P10 promoter and PH promoter.Finally,the constructing eukaryotic expression plasmid pFastBacTM dual-HA1-mChIL-18 was transposited into Bacterium Coli DH10Bac,which was based on next step insect cell's transfection.The construction of this recombinant plasmid was benefit to investigate the effect of mChIL-18's immunologic enhancement and study a new AIV recombinant vaccine.