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In this study, extraction of nucleic acid from a Guangdong duck origin avian influenza virus A/duck/Guangndong/2003 (H5N1) , using the PCR method , hemagglutinin ( HA ) gene fragment about 1800 bp , recycling target fragments obtained by PCR and connected to the HA gene cloning vector PMD18-T , a new plasmid , named PMD18 - T- HA sequencing results showed that the full-length sequence 1851bp, contains a complete open reading frame of the HA gene and the start codon and stop codon . The the plasmid PMD18-T-HA pDsRed2 with restriction endonuclease and the HA gene the fragment with pDsRed2 plasmid vector plasmid named PHD1 . Meanwhile, from the the Marek virus ( MDV) 1 Standard strains extracted the MDV nucleic acid , the use of the PCR method to obtain US1 that US2 is fragment, the cloning vector pMD18- T specification , US1, US2 are respectively connected to the cloning vector pMD18- T , obtained new plasmid PMD18 - T -US1 PMD18 - T -US2 . Positive clones were sequenced and analyzed , and the results show that the obtained US1 full-length 1147bp, US2 total length of 1246bp . After the plasmid PMD18-T-US1 and PUC19 digested with the restriction enzyme digestion, then US1 connected to the multicloning site of plasmid PUC19 , to form a new plasmid was named PUCF1 successfully constructed , the plasmid pMD18- the T- US2 in the the US2 gene fragment by restriction enzyme digestion , and connected to the plasmid PUCF1 , to form a new plasmid was named PUCF120 . Plasmid PHD1 template to design primers to amplify the purpose of containing HA reading frame and fluorescent red chromogenic gene fragment and cloned into the vector pGEM -Teasy , then objective fragment by restriction endonucleases , and the newly constructed plasmid the digested PUCF12 with T4 ligase to connect successfully build the final plasmid RHAF12 . In summary, this study through a series of PCR recovery, connected transformation test method, the resultant HA, US1, US2 gene and vector PUC19, pDsRed2 connection to construct the target plasmid , avian influenza for future genetic engineering vaccine research laid a solid foundation , but also provide a strong scientific basis for the prevention and control of avian influenza in China .
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