论文部分内容阅读
目的:构建含有甲型流感病毒M2基因与GM-CSF基因的DNA疫苗双表达载体,为研究甲型通用流感疫苗奠定基础。方法:将甲型流感病毒(H1N1)接种鸡胚后收获尿囊液,提取流感病毒总RNA,RT-PCR法扩增M2基因;将微小病毒内部核糖体进入位点(IRES)基因插入到质粒pVAXⅠ多克隆位点,再将M2基因和GM-CSF基因依次克隆到IRES的上、下游多克隆位点,构建出甲型流感病毒双表达载体pMIG,酶切鉴定后测序;脂质体法转染COS7细胞并检测目的蛋白的表达。结果:成功扩增出M2基因(约300bp)、IRES基因(约580bp)和GM-CSF基因(约400bp),酶切鉴定结果表明构建出甲型流感病毒双表达载体pMIG,Western blotting检测证明流感病毒M2蛋白的表达。结论:成功构建出流感病毒DNA疫苗双表达载体pMIG。
OBJECTIVE: To construct a DNA vaccine double expression vector containing M2 gene and GM-CSF gene of influenza virus A, which lays the foundation for the study of influenza A vaccine. Methods: The allantoic fluid was harvested after inoculated with chicken embryo of type A (H1N1) and the total RNA of influenza virus was extracted. The M2 gene was amplified by RT-PCR. The inserted internal gene of microbe virus (IRES) pVAX Ⅰ multiple cloning site, and then M2 gene and GM-CSF gene were cloned into the IRES upstream and downstream multiple cloning site, the construction of influenza A virus double expression vector pMIG, restriction enzyme digestion and sequencing; liposome method COS7 cells were stained and the expression of the protein of interest detected. Results: The M2 gene (about 300bp), the IRES gene (about 580bp) and the GM-CSF gene (about 400bp) were amplified successfully. The restriction endonuclease digestion results showed that influenza A virus double expression vector pMIG was constructed and confirmed by Western blotting Virus M2 protein expression. Conclusion: The double DNA vaccine vector pMIG of influenza virus was successfully constructed.