噬菌体裂解酶Lysep3的毕赤酵母重组表达和活性分析

doi:10.16431/j.cnki.1671-7236.2020.04.006

摘要/Abstract

摘要： 试验以突破噬菌体裂解酶Lysep3应用瓶颈为切入点，采用酵母表达系统对其进行重组表达以期降低生产成本，同时初步探究其体外抗菌活性和安全性，以期为临床试验提供数据参考。根据毕赤酵母密码子偏爱性优化并合成Lysep3基因，将其连接至表达载体pPICZαA。经菌落PCR和测序验证后，将线性化重组载体pPICZαA-Lysep3电转化巴斯德毕赤酵母Pichia pastoris X-33。重组酵母转化子经菌落PCR和摇瓶水平甲醇诱导重组蛋白初筛后，在5 L发酵罐中高密度发酵，发酵上清液经His-trap HPGE纯化柱纯化。通过抑菌试验和浊度试验进行rLysep3的抑菌活性分析，通过测定rLysep3对巨噬细胞RAW264.7细胞毒性和小鼠血红细胞溶血性进行rLysep3的安全性分析。结果显示，重组菌在5 L发酵罐中甲醇诱导120 h后Lysep3的表达量达749.2 mg/L。抑菌活性分析发现，rLysep3对肠炎沙门氏菌、金黄色葡萄球菌、猪葡萄球菌及无乳链球菌均具有抑菌活性，且rLysep3可与EDTA联合作用使肠炎沙门氏菌菌落数下降1.6~1.7个数量级。此外，细胞毒性试验发现，rLysep3在1 024 μg/mL浓度下细胞存活率仍可达78.75%；溶血性分析显示，1 024 μg/mL浓度下溶血率仅为1.58%。上述结果证明，通过毕赤酵母表达系统实现了Lysep3的重组表达，rLysep3具有体外抑菌活性，且具低细胞毒性和低溶血性。从抗菌活性、产业化制备及安全性等方面初步显示出rLysep3具有开发为临床治疗禽肠炎沙门氏菌感染的新型抗菌药物的潜力。

关键词: 噬菌体裂解酶Lysep3; 毕赤酵母; 重组表达; 肠炎沙门氏菌; 抗菌活性

Abstract: In order to break through the application bottleneck of phage lyase Lysep3,Lysep3 was recombinantly expressed using yeast expression system for its low-cost production,and in vitro activity and safety of Lysep3 were explored preliminarily to provide data reference for clinical experiments.Lysep3 gene was optimized according to the codon bias of Pichia pastoris and synthesized,and ligated into the expression vector pPICZαA.After verification of colony PCR and sequencing,the linearized recombinant vector pPICZαA-Lysep3 was electro transformed into P.pastoris X-33.After colony PCR and recombinant protein screening at shake flask level by methanol induction,recombinant yeast were fermented with high-density in 5 L fermenter,and fermentation supernatant was purified by His-trap HPGE purification column.The antibacterial activity of rLysep3 was analyzed by inhibition zone test and turbidity test.The safety of rLysep3 was analyzed by cytotoxicity of rLysep3 against macrophage RAW264.7 and hemolytic activity of rLysep3 against mouse red blood cells were assayed for its safety evaluation.In 5 L fermenter,Lysep3 was heterologously expressed in P.pastoris and its expression level reached 749.2 mg/L.The activity analysis of rLysep3 showed that it had antibacterial activity against Salmonella enteritidis,Staphylococcus aureus,Staphylococcus hyicus and Streptococcus agalactiae,and the number of S.enteritidis colonies was reduced by 1.6 to 1.7 orders of magnitude through the combination of rLysep3 and EDTA.In addition,cytotoxicity assays showed that rLysep3 still had a cell viability of 78.75% at a concentration of 1 024 μg/mL;hemolytic analysis showed rLysep3 only had a hemolysis rate of 1.58% at a concentration of 1 024 μg/mL.The above results demonstrated that the recombinant expression of Lysep3 could be achieved by the P.pastoris expression system,and rLysep3 had in vitro antibacterial activity,low cytotoxicity and low hemolysis.From the aspects of antibacterial activity,industrial preparation and safety,it showed that rLysep3 had the potential to develop a new antibacterial drug for the clinical treatment of S.enteritidis infection.

Key words: phage lyase Lysep3; Pichia pastoris; recombinant expression; Salmonella enteritidis; antibacterial activity

中图分类号:

S859.79⁺7

闫伟, 杨娜, 毛若雨, 王秀敏, 郝娅, 马炫炫, 滕达, 王建华. 噬菌体裂解酶Lysep3的毕赤酵母重组表达和活性分析[J]. 中国畜牧兽医, 2020, 47(4): 1018-1027.

YAN Wei, YANG Na, MAO Ruoyu, WANG Xiumin, HAO Ya, MA Xuanxuan, TENG Da, WANG Jianhua. Recombinant Expression and Activity Analysis of Phage Lyase Lysep3[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(4): 1018-1027.

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