论文部分内容阅读
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,通过引入内部核糖体进入位点序列(IRES),构建共表达H5N1亚型禽流感病毒膜蛋白基因M1和HA的重组腺病毒。PCR扩增禽流感病毒H5N1亚型M1和HA基因的全长可读框片段,先后亚克隆入pStar载体,然后扩增M1-IRES-HA片段并将其插入穿梭载体pShuttle-CMV,再与pAd-Easy载体在BJ5183菌中通过同源重组产生重组腺病毒载体,转化293细胞,包装出重组腺病毒Ad-M1/HA。将Ad-M1/HA感染293细胞,可观察到明显细胞病变效应,用免疫荧光及Western-blot方法均检测到M1和HA基因的表达。共表达M1和HA双基因的重组腺病毒的成功构建为开发新型重组腺病毒流感疫苗奠定了基础。
A H5N1 subtype avian influenza virus (A / Anhui / 1/2005) isolated from China was used as the research object to construct a co-expression membrane protein of H5N1 avian influenza virus by introducing an internal ribosome entry site sequence (IRES) Recombinant adenovirus with genes M1 and HA. The full-length open reading frame fragment of M1 and HA genes of H5N1 subtype of avian influenza virus was amplified by PCR, subcloned into pStar vector, and then amplified M1-IRES-HA fragment and inserted into the shuttle vector pShuttle-CMV, -Easy vector The recombinant adenovirus vector was generated by homologous recombination in BJ5183 strain, transformed into 293 cells and packaged recombinant adenovirus Ad-M1 / HA. Infect 293 cells with Ad-M1 / HA, obvious cytopathic effect was observed. The expression of M1 and HA genes were detected by immunofluorescence and Western-blot. The successful construction of the recombinant adenovirus co-expressing the M1 and HA double genes laid the foundation for the development of a new recombinant adenovirus influenza vaccine.