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

Abstract

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

CLC Number:

S859.79⁺7

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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Recombinant Expression and Activity Analysis of Phage Lyase Lysep3

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