摘要
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 the DNA vaccine coexpression vector containing M2 gene of influenza A virus and GM-CSF gene, which provides a basis for exploring broad-spectrum vaccine of influenza A virus. Methods Analogous strain of influenza A virus （H1N1） was inoculated by chicken and alantoic fluid was collected, the total RNA of influenza A virus was extracted, M2 gene was amplified by RT-PCR. Internal ribosome entry site （IRES） gene of minute virus was inserted into the multiple cloning sites （MCS） of the plasmid pVAX I , then M2 gene and GM-CSF gene were in turns cloned into the MCS of the advanced and backward MCS of IRES for constructing pMIG-the coexpression vector of influenza A virus and sequencing after enzymetomy identification. Then the recombinant plasmid pMIG was transfected into COS7 cell by liposome method and the expression of the target protein was detected. Results M2 gene （300 bp）, IRES gene （580 bp） and GM-CSF gene （400 bp） were successfully amplified, the result of enzymetomy identification indicated that the coexpression vector pMIG of influenza A virus was constructed, Western blotting testified that M2 protein of influenza A virus was expressed. Conclusion The coexpression vector pMIG of influenza A virus is successfully constructed.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2008年第1期65-68,共4页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅基金资助课题(200505184)
