Rbc t a cloning vector

The partial sequence of the PmVRP15 cDNA was initially obtained from the SSH library of WSSV-challenged P. monodon hemocytes, and then extended by 5′ RACE. The hemolymph of ∼20 g body weight shrimps was drawn from the ventral sinus using a sterile 1-mL syringe with 150 μL of 10% (w/v) sodium citrate solution. The hemolymph was immediately centrifuged at 5000×g for 5 minutes at 4 °C to separate the hemocytes (pellet) from the plasma (supernatant). Total RNA was isolated from the hemocytes using the TRI Reagent (Molecular Research Center) according to the manufacturer's protocol. A full-length cDNA of PmVRP15 was determined using The SMART RACE cDNA Amplification Kit (Clontech) and the GSP-RACE primer (Table 1), according to the manufacturer's instruction. The RACE product was purified using a NucleoSpin Extract II kit (Clontech) according to manufacturer's protocol, and cloned into the RBC T&A Cloning Vector (RBC Bioscience). Then, the recombinant plasmid was transformed into Escherichia coli DH5α competent cells (RBC Bioscience). The positive clones were commercially sequenced by Macrogen INC., South Korea. The nucleotide sequences of SSH clone and RACE fragment were then assembled and searched against the NCBI database.

The cDNA of K148 NDV was divided into six fragments with ~300 nucleotides of overlapping regions between adjacent fragments (Figure 1). All six fragments were amplified with six sets of gene-specific primers and cloned into the RBC TA cloning vector (RBC Bioscience, Taipei, Taiwan; Supplementary Table 1). Each fragment was sequentially inserted into the pBluescript vector using the In-Fusion® PCR cloning kit (Clontech, Mountain View, CA). The pfuUltra^TM II fusion HS DNA polymerase (Stratagene, La Jolla, CA) was used to amplify the in-fusion fragments and linearizing vectors. The vector plasmid was linearized before each step of the fragment in-fusion. Nucleoprotein (NP), polymerase (P), and large polymerase (L) genes were cloned into an expression plasmid to construct the supporting plasmids. The primers used for vector linearization and in-fusion are listed in Supplementary Table 2. After construction of the full-length cDNA clone of the K148 virus (pK148), pK148 was transformed into HIT-DH5α competent cells (RBC Bioscience) at 37°C for 18 h and purified using the PureLink™ HiPure plasmid midiprep kit (Thermo Fisher Scientific).

pK148 was linearized to insert the HA gene of ES2 HPAIV virus and an additional cDNA fragment containing the untranslated region (UTR), including the gene end, and gene start (GS) signals, between polymerase gene (P) and matrix (M) gene of the full genome K148 cDNA clone. The additional UTR and cDNA of ES2 HA gene were amplified by PCR and cloned into RBC TA cloning Vector (RBC Bioscience, Taipei, Taiwan). The multibasic cleavage site of the HA gene (PLRERRRKR/GLF) was replaced by the cleavage site of LPAIV (PLRERR/GLF). To enhance the expression of ES2 HA gene, Kozak sequence (ACC) was inserted at the upstream of HA gene by using Muta-direct^TM site-directed Mutagenesis kit (iNtRON Biotechnology, Korea). The cDNA of UTR and the HA gene were ligated and inserted between P and M genes of pK148 by infusion PCR described above, forming pK148/ES2-HA clone. The pK148/ES2-HA clone was amplified and purified as described above.