人高致病性禽流感病毒H5N1安徽株HA、M2蛋白双顺反子表达载体的构建及表达研究

Construction of Bicistronic Eukaryotic Expression Vector Containing HA and M2 Genes Derived from High Pathogenic Avian Influenza Virus(HPAI)H5N1(Anhui strain)and Its Efficient Expression in Mammalian Cells

将我国分离的人H5N1亚型禽流感病毒A/Anhui/1/2005作为研究对象,扩增其HA和M2基因片段并克隆至DNA疫苗表达载体pVRC中,构建成真核表达质粒。为提高HA的表达量,按照人偏爱密码子将HA基因进行优化改造,经全基因合成后插入真核表达载体pVRC,以β-actin蛋白为内参比证明了优化后的HA蛋白表达效果明显提高。将M2基因和优化后的HA基因共同克隆入双顺反子表达载体pIRES中,获得同时表达HA或M2的双顺反子真核表达质粒;通过Western blot和间接免疫荧光检测方法,确认构建的重组质粒在真核细胞中成功地表达了目的蛋白HA和M2。通过上述结果为进一步开展人高致病性禽流感病毒安徽株HA和M2基因的功能与致病性研究及使用表达HA和M2蛋白进行新型人用禽流感双价疫苗研发奠定基础。

HA and M2 genes derived from human highly pathogenic avian influenza H5N1 virus （A/Anhui/ 1/2005） isolated from China, were amplified and cloned into the DNA vaccine expression vector pVRC. In order to improve the expression of hemagglutinin, the human codon usage preference was made and the whole length of HA gene of HSNI（A/Anhui/1/2005） influenza virus was synthesized,named HA/YH/K, and inserted to pVRC vector, the expression of HA/YH/K protein in eukaryotic cells was significantly improved according to internal control of actin protein. Furthermore, the M2 and HA/YH/K genes were cloned into bicistronic eukaryotic expressing vector pIRES to yield the recombinant plasmid pIRES-HA/ YH/K-M2/YS/K, which could expressed HA and M2 protein simultaneously by transfection of one plasmid. Western blot and IFA showed that the recombinant pIRES-HA/YH/K-M2/YS/K plasmid was successfully expressed in several mammalian cells （Hela, MDCK and 293FT）. The above results may help to identify the function and pathogenic mechanism of HA, M2 genes derived from HPAI H5N1 （Anhui strain）and pave a way for the development of novel bivalent vaccines against human highly pathogenic avian influenza virus and for preparedness for influenza pandemic.