Expression of Procine Lactoferrin N-lobe(PLF-N) in Pichia pastoris and Detection of its Antimicrobial Activity

According to the aminoacid sequences of porcine lactoferrin N-lobe(PLF-N)and partiality codon of Pichia pastoris,the porcine lactoferrin N-lobe gene was designed and synthesized.The modified porcine lactoferrin N-lobe gene was cloned into the pGAPZα-A vector to construct the recombinant expression vector pGAPZα-A-PLF-N.The Avr Ⅱ linearized plasmid pGAPZα-A-PLF-N was transformed into Pichia pastors SMD1168 by electroporation.The transformants were identified by PCR,screened with Zeocin,and multiply-copy colonies were harvested,in which PLF-N gene was verified to inserted into yeast chromosome stably.Under the control of the promoter GAP,the PLF-N recombinant protein with a molecular weight of 38 ku was expressed in supernatant.Up to 364 μg/mL PLF-N protein was produced in culture medium.Agrose diffusion assay showed that PLF-N had broad-spectrum antibacterial abilities not only to gram-negative bacteria but also to gram-positive bacteria and with MIC of 12.5,25 μg/mL against E.coli DH5α and Staphylococcus aureus,respectively.