Eukaryotic Expression Of HA And M2 Genes Of H5N1 Subtype High Pathogenic Avian Influenza Virus

Two pairs of clone primers was designed and synthesized based on HA and M2 genes sequences of known H5N1 subtype human lfighly pathogenic avian influenza virus.The HA and M2 genes were amplified by using specific primers and PCR.The amplified DNA fragment was cloned into pMD18-T vector for sequencing analysis and restriction enzyme digestion.The HA and M2 genes was cloned into pVRC eukaryotic expressing vector to yield the recombinant plasnfids pVRC-HA/YS/K and pVRC-M2/YS/K.Then these recombinant plasmids were transfected into MDCK cell or 293FT cell with Lipofectamine 2000 Transfection Reagent after identification by restriction enzyme analysis and sequencing analysis.We verified the expression with fluorescence analysis and western blot analysis.We optimized the codon usage of wild type H5N1 AIV HA gene according to the codon bias of mammalian cell and synthesized the full-length optimized HA gene named HA/YH/K and wild type HA gene named HA/YS/K.The HA/YH/K gene was cloned into pVRC vector to yield the recombinant plasmid pVRC-HA/YH/K.Then the recombinant plasmid was transfected into MDCK cell or 293FT cell with Lipofectamine 2000 Transfection Reagent after identification by restriction enzyme analysis and sequencing analysis.We verified the expression with fluorescence analysis and western blot analysis.We will analysis HA protein level under the same condition according to internal control of actin protein.The HA/YH/K and M2/YS/K genes were cloned into pIPES eukaryotic expressing vector to yield the recombinant plasmid plRES-HAfYH/K-M2/YS/K.Then the recombinant plasmid was transfected into 293FT cell with Lipofectamine 2000 Transfection Reagent after identification by restriction enzyme analysis and sequencing analysis.We verified the expression with fluorescence analysis and western blot analysis.The HA/YS/K and M2/YS/K genes were cloned successfully and pVRC-HA/YH/K,pVRC-HA/YS/K,pVRC-M2/YS/K were cloned successfully,the result of restriction enzyme digestion and DNA sequencing was correct.The eukaryotic expressing vector pVRC-HA/YH/K, pVRC-HA/YS/K,pVRC-M2/YS/K and pIRES-HA/YH/K-M2/YS/K were cloned successfully and they could express HA and M2 protein correctly,in accordance with related documents.The results demonstrated the expression of HA protein on the HA/YH/K has successfully improved protein expression than the HA/YS/K. the eukarytic expressing vector pIRES-HA/YH/K-M2/YS/K can coexpressed HA and M2 proteins and the protein level of HA and M2 protein on the coexpression were less than targeted protein signal expression according to internal control of actin protein.The eukaryotic expressing vector containing HA and M2 genes of H5N1 subtype human highly pathogenic avian influenza virus,transfer it into eukar-yotic cell and detect the expression of target protein,and to provide a basis for illustration of the function and pathogenic mechanisms of HA and M2 genes,further more,for the development of novel vaccines against human H5N1 influenza virus.