猪乳铁蛋白N叶(PLF-N)在毕赤酵母中的表达及抑菌活性鉴定

根据猪乳铁蛋白N叶(PLF-N)的蛋白序列和毕赤酵母(Pichia pastoris)密码子的偏嗜性,通过全基因合成法合成改造后的PLF-N基因片段,将其克隆到pGAPZα-A质粒中构建分泌型重组酵母表达载体 pGAPZα-A- PLF。pGAPZα-A- PLF 通过限制性内切酶Avr Ⅱ酶切线性化后,经电穿孔法转入毕赤酵母细胞SMD1168内。PCR检测结果发现,PLF-N基因与毕赤酵母染色体稳定结合,Zeocin抗性筛选,得到高拷贝转化子。在GAP启动子调控下,PLF-N重组蛋白获得分泌表达,其分子质量约为38 ku,上清表达量约为364 μg/mL。琼脂糖孔穴扩散法检测结果显示,该表达产物对革兰氏阴性菌和革兰氏阳性菌均具有较好的抑菌活性,且对大肠杆菌和金黄色葡萄球菌最小抑菌浓度(MIC)分别为12.5、25.0 μg/mL。

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.