[Expressing of porcine beta-defensin-2 mature peptide in the yeast].

OBJECTIVE

To construct the recombinant Pichia pastoris KM71 in which Porcine beta-defensin-2(pBD-2) mature peptide could be stably expressed and the antimicrobial effect was evaluated.

METHOD

According to the requirement of yeast codon bias for protein expression, three pairs of primers were designed for development of SOE-PCR to amplify the genes coding pBD-2 mature peptide. The resultant genes were cloned into pPIC9k and pPIC9k-Gultathione S Transferase (GST) vectors to yield the recombinant expressing vector, pPIC9K-pBD-2 and pPIC9k-GST-pBD-2. The two recombinant plasmids were linearized, followed by transformation with Pichia pastoris KM71 cells to produce the positive clones of recombinant yeasts. The expression conditions were continuously optimized to produce mature peptide of pBD-2.

RESULT

Gene fragments for GST-fusion PBD-2 and pBD-2 alone were amplified and integrated into genomic chromosome of the yeast KM71, respectively, and the recombinant yeasts were obtained; GST-fusion pBD-2 peptide and singular pBD-2 peptide were successfully expressed. The mature peptide of pBD-2 showed a certain inhibition effect on the growth of the Salmonella choleraesuis C500- strain.

CONCLUSION

The pBD-2 was expressed successfully and casted a light for massive production of the pig defensin, a new antimicrobial agent.