H5N1亚型流感病毒M2与NA基因真核表达载体的构建与鉴定

Construction of vectors expressing M2 and NA genes of H5N1 influenza virus

目的 构建表达H5N1亚型流感病毒M2和NA基因的多种真核表达载体.方法 以我国分离的首株人H5N1亚型禽流感病毒（A/Anhui/1/2005）作为研究对象,PCR扩增全长M2与NA基因.将M2基因克隆入真核表达载体pStar的IRES上游或下游的MCS,构建重组pStar-M2/和pStar-/M2.将NA基因克隆人pStar载体IRES上游或下游的MCS,构建重组pStar-NA/和pStar-/NA.将M2和NA基因先后克隆到pStar载体IRES序列的上游和下游MCS,分别构建双基因共表达重组pStar-M2/NA和pStar-NA/M2.将重组质粒转染293细胞,使用IFA方法 检测各重组质粒相应外源基因的表达.结果 通过酶切鉴定,各重组质粒构建正确.将其转染293细胞后,使用IFA方法 检测到了各重组质粒中相应外源基因的表达.结论 构建了多种表达H5NI亚型流感病毒M2和（或）NA基因的真核表达载体,为研究开发流感DNA疫苗奠定了基础.

Objective To construct vectors expressing M2 and NA genes of H5N1 influenza virus. Methods Based on the human HSN1 avian influenza virus （ A/Anhui/1/2005） isolated in china, M2 and NA genes were amplified by PCR. M2 or NA gene was subcloned into pStar vector to construct recombinant pStar-M2/, pStar-/M2, pStar-NA/and pStar-NA/. Furthermore, both of the M2 and NA genes were subcloned into pStar to construct two genes co-expressing recombinant pStar-M2/NA and pStar-NA/M2. Expression of the genes were detected by IFA after transfection of 293 cells with the recombinant plasmids. Results Recombinant plasmids were constructed and identified by restriction endonuclease digestion. Expression of the genes cloned into the recombinant plasmids was confirmed by IFA. Conclusion Recombinant plasmids expressing M2 and/or NA genes of H5N1 influenza virus were constructed, which provided basis for development of influenza DNA vaccine.