鸡源抗菌肽Folicidin-3在毕赤酵母中的分泌表达及其生物学活性鉴定

Expression of the Chicken Antibacterial Peptide Fowlicidin-3 in Pichia Pastoris and Identification of Biological Activity

【目的】研究鸡源重组抗菌肽Fowlicidin-3的生物学特性,并为重组抗菌肽在临床上的应用奠定基础。【方法】根据抗菌肽数据库中Fowlicidin-3的氨基酸序列,选用毕赤酵母的偏嗜密码子,设计抗菌肽的基因,通过SOE法合成得到全长为114bp的Fowlicidin-3基因。将Fowlicidin-3基因克隆入pPICZα-A质粒,构建分泌型重组酵母表达载体pPICZα-A-F。将线性化重组表达载体pPICZα-A-F通过电击法转入毕赤酵母宿主菌X-33中,构建分泌型重组酵母表达菌株。【结果】获得分泌型重组酵母表达菌株,Fowlicidin-3多肽表达量达到170mg·L-1,对致病性大肠杆菌K99和鸡白痢沙门氏菌显示出较好的抑制活性,抑菌圈直径分别为1.9cm和2.2cm。优化表达条件结果显示,转接到BMMY培养基中(pH6.0),每24h补加2%甲醇,29℃培养72h后,Fowlicidin-3抗菌肽获得高效表达。【结论】成功构建分泌型重组酵母表达菌株,并获得了具备高效表达能力和较好生物学活性的抗菌肽。

【Objective】 The biological property of the recombinant antibacterial peptides Fowlicidin-3 and its application in animal husbandry were studied. 【Method】 According to the amino acid sequences of Fowlicidin-3 as described in the antibacterial peptide database, using the preferential condon of P. pastoris, the antibacterial peptide Fowlicidin-3 gene 114 bp in length was designed and synthesized by SOE PCR. The antibacterial peptide gene was cloned into the pPICZα-A vector to construct the recombinant expression vector pPICZα-A-F. The linearized plasmid pPICZα-A-F was transformed into P. pastoris X-33 by electroporation to construct the recombinant expression yeast strain. 【Result】 Fowlicidin-3 was successfully expressed and the concentration of the secreted antibacterial peptides reached up to 170 mg·L-1. Agrose diffusion assay showed that antibacterial peptide Fowlicidin-3 exhibited strong antibacterial abilities to E. coli K99 and Salmonella pullorum, the diameter of inhibition zone to E. coli K99 and Salmonella pullorum were 1.9 cm and 2.2 cm, respectively. The result also showed that the recombinant antibacterial peptides Fowlicidin-3 could be expressed and obtained with high level while the pH of the culturing BMMY medium was 6.0, the yeast was cultured under 29℃ for 72 hours and 2% methanol was supplemented into the medium. 【Conclusion】 The high level expression of antibacterial peptide Fowlicidin-3 with great biological activities was successfully constructed in yeast strain P. pastoris in this research.