Construction and application of H7N9 avian influenza virus gene recombinant plasmid pGEM-HA-NA-M

Objective To construct a combinant plasmid of full length HA/NA/M gene of H7N9 avian influenza virus and to provide quantitative reference for pathogen detection.Methods According to specific sequence of HA/NA/M gene of H7N9 avian influenza virus,the primers were designed and synthesized.Total RNA extracted from H7N9 avian influenza virus and the cDNA of of HA/NA/M were cloned by reverse transcription PCR(RT-PCR)and inserted into pGEM-T easy vector after three times of restriction enzyme assay.The linearized plasmids were used to transcript RNA in vitro by T7 RNA polymerases,then the products were purified and diluted to a series of standard concentrations of cRNA which was used as standard quantitative template of real-time fluorescence quantitative RT-PCR method.Results The amplified fragment by RT-PCR was of expected size and its sequence was in concordance with that published on GenBank.The cRNA including full-length HA/NA/M was obtained by in vitro transcription with the recombinant plasmid and the mass concentration was 399.5 ng/μl.The cRNA were diluted to precise quantification copy number,which were proved by real-time RT-PCR amplification.Conclusion The combinant plasmid pGEM-HA-NA-M was constructed successfully and in vitro transcription products of the plasmid can be used as a quantitative reference for the rapid detection of nucleic acid of H7N9 avian influenza virus.