H5N1亚型禽流感病毒ns1基因修饰

Modification of ns1 Gene of H5N1 Avian Influenza Virus

目的:对H5N1亚型禽流感病毒株(A Qa ST85201)的ns1基因进行修饰并构建ns1基因的真核表达载体。方法:用RT PCR方法扩增A Qa ST85201的ns1基因,之后以扩增产物为模板,采用多重PCR技术对ns1基因进行修饰,将缺失的15个核苷酸片段插入ns1基因中,并将其克隆到真核表达载体,构建pcDNA3.1ns1重组质粒。结果:RT PCR产物测序显示,A Qa ST85201ns1基因开放阅读框全长678bp,编码225个氨基酸。构建的重组质粒经酶切鉴定和序列测定与预期相一致。结论:成功将缺失的15个核苷酸片段引入A Qa ST85201的ns1基因中,重组质粒的构建为NS1蛋白功能研究奠定了基础。

Objective:To modify the ns1 gene of avian influenza virus H5N1 subtype(A/Qa/ST/852/01)and to construct the recombinant expression vector. Methods:The ns1 gene of A/Qa/ST/852/01 was amplified by RT-PCR and a deletion of 15 nucleotides (acid) was inserted into ns1 gene by multiply PCR, then cloned into expression vector pcDNA 3.1(+).Results:The sequence analysis demonstrated that the ns1 gene of A/Qa/ST/852/01 contained one open rea-(ding) frame of 678 bp, which encoded polypeptides of 225 amino acids. The recombinant plasmid of pcDNA 3.1/ns1 was digested with restriction endonuclease and DNA sequence analysis. Conclusion:The deletion of 15 nucleotides acid is successfully inserted into the ns1 gene of A/Qa/ST/852/01,and the construction of the eukaryotic expression vector provides a foundation for further studies on the function of NS1 protein.