Pfastbac ht c plasmid

The H5N2 (A/duck/Yunlin/04) full-length NP was amplified by reverse transcription-polymerase chain reaction (PCR) with forward primer NP-F: 5′-ATGGCGTCTCAAGGCACCAAAC-3′ and reverse primer NP-R: 5′-TTAATTGTCATACTCCTCTGCATTGTC-3′. The NP full-length gene was cut from the T&A Cloning Vector (Yeastern Biotech, Taiwan) and subcloned in a frame into the corresponding pFastBac-HT-C plasmid (Invitrogen, USA). Additionally, a 6 histidine residue was designed to tag on the 3′ end of the NP by engineering the Spe I site embedded reverse primer NP-HisSpel-R: 5′-AGCACTAGTTCAGTGGTGGTGGTGGT-3′ in company with the EcoRI site embedded forward primer NP-EcoR-F: 5′-AGGAATTCGAATGGCGTCTCAAGGCAC-3′ to obtain the PCR product and used to construct the rNP vector. Subsequently, the recombinant vector was transposed into the baculovirus (Autographa californica) genome to form NP pFastBac recombinant bacmids. Positive recombinant bacmids were used to transfect an insect cell line (Spodofera frugiperda, Sf9) for viral particle formation. All procedures for viral particle production were performed according to the manufacturer's manual (Bac-to-Bac; Invitrogen). Recombinant NP from baculovirus-infected Sf9 cells was obtained, purified by Ni-NTA Agarose (Invitrogen), and used as an antigen for WB analysis.