The PCR was carried out under a cycle protocol of pre-denaturation at 94°C for 4 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 61°C for 30 sec, and extension at 72°C for 45 sec, and a final elongation at 72°C for 10 min. The PCR products were then purified on 1.5% agarose gel and the DNA bands were extracted using E.Z.N.A. Gel Extraction Kit (Omega Biotek).
The lhb fragment was digested with double restriction endonuclease EcoRI and HindIII, and cloned into corresponding double restriction endonuclease-digested pFastBacDual bacmid transfer vector (Invitrogen) to generate the construct of gpFast-lhb, and then the cga cDNA treated with Nhe I and Kpn I double restriction endonuclease digestion was cloned into the constructed gpFast-lhb, which had been double restriction endonuclease digested. This vector contained a mini-Tn7 element, an E. coli origin of replication, and an amplicillin marker.