Complementary DNA Cloning and Construction of Recombinant Baculovirus Transfer Vector
In the present study, the ORF cDNAs encoding grouper cga and lhb were subcloned using RT-PCR. The recombinant transfer vector was constructed and digested by double restriction endonuclease digestion analysis. Several aim fragments were released in double restriction endonuclease digestion analysis (EcoRV + EcoRI; EcoRI + HindIII; HindIII + Nhe I; Nhe I + EcoRV; Kpn I and EcoR I; HindIII and EcoRV), which were used to prove the correct construction of the recombinant transfer vector (Fig. 1).
PCR and Southern Blot Analysis of Recombinant Bacmid-lh
The bacmid DNA was >135 kb. It was difficult to verify the insertion of the specific gene with the typical restriction endonuclease digestion analysis. Therefore, PCR was used to confirm transposition of the target gene to the bacmid. The pUC/M13 amplification primers were directed at sequences on either side of the miniattTn7 site within the lacZa-comple-mentation region of the bacmid. The PCR product of successful transposition was about 3361 bp, while the PCR fragment without transposition was about 273 bp (Fig. 2C).
FIG. 1. Construction (A) and the restriction enzyme digestion (B) of recombinant transfer vector pFast-lh. M, 1 kb DNA ladder marker;1, plasmid pFast-lh digested with EcoRV and EcoRI;2, pFast-lh digested with EcoRI and HindIII; 3, pFast-lh digested with HindIII and Nhe I;4, pFast-lh digested with Nhe I and EcoRV; 5, pFast-lh digested with Kpn I and EcoR I; 6, pFast-lh digested with HindIII and EcoRV.
FIG. 2. Bacmid DNA (A), Southern blot (B), and PCR (C) analysis of bacmid-lh DNA. A, B) M, k DNA/H/ndIII marker;1, no transposition bacmid DNA;2,3,4,5, transposition bacmid-lh DNA. C) M, 1 kb DNA ladder marker;1, no recombinant bacmid;2, 3, 4, 5 recombinant bacmid-lh DNA.