天蚕素A和溶菌酶杂合肽在毕赤酵母中重组表达及其活性分析

doi:10.16431/j.cnki.1671-7236.2023.12.025

Abstract

Abstract: 【Objective】 This experiment aimed to produce a novel cecropin A and lysozyme fusion peptide (CecA-Lyz) using Pichia pastoris and to explore its antibacterial activity.【Method】 The gene encoding the fusion peptide CecA-Lyz was optimized according to the codon preference of Pichia pastoris and the physicochemical properties of CecA-Lyz was analyzed.The optimized CecA-Lyz gene was inserted into the plasmid pPIC9K-HSA to construct the recombinant plasmid pPIC9K-HSA-CecA-Lyz,which contained a 6×His tag and HSA DⅠ&DⅡ region at N-terminal.The recombinant plasmid was linearized using PmeⅠ and electrotransformed into the Pichia pastoris GS115 competent cell.The recombinant strain of high-level expressed CecA-Lyz protein was selected in YPD+G418 plate.Moreover,the optimum induction conditions of recombinant CecA-Lyz protein,including the methanol concentration and induction time were explored,and the antibacterial activity of recombinant CecA-Lyz was verified.【Result】 After codon optimization,the GC content of the CecA-Lyz gene was 48.9%,and the codon adaptation index (CAI) was 0.87,which was suitable for secretory expression in Pichia pastoris.The expected molecular weight of CecA-Lyz protein was 20.8 ku,with a theoretical isoelectric point of 9.62 and relatively stable physicochemical properties.Moreover,the optimized CecA-Lyz gene was successfully contructed into expression plamid and produced recombinant CecA-Lyz fusion protein in Pichia pastoris.The optimal methanol concentration for the induction of recombinant CecA-Lyz protein was 2.5% and the optimal induction time was 72 h.In addition,the recombinant fusion peptide without N-terminal HSA DⅠ&DⅡ was obtained by TEV digestion.The recombinant CecA-Lyz was initially shown to have antibacterial activity against Escherichia coli by plate counting and Oxford cup methods,including inhibition of the growth of drug-resistant Escherichia coli isolated from duck farms,with the inhibitory diameter up to 1.45 cm,but no significant inhibition against Salmonella and Staphylococcus.【Conclusion】 The recombinant CecA-Lyz fusion peptide could be stably produced in Pichia pastoris to obtain the fusion peptide without HSA tag and with the effect of inhibiting growth of Escherichia coli,which provided research ideas and references for further development of antimicrobial peptides.

Key words: antimicrobial peptide; Pichia pastoris; fusion protein; hybrid peptide

CLC Number:

S859.79⁺7

BUAYISHAM Kuerban, WANG Yixin, YANG Chengxi, GENG Zijian, XU Yingqing, LUO Gang. Recombinant Expression of Cecropin A and Lysozyme Heteropeptide in Pichia pastoris and Its Activity Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 5032-5042.

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Recombinant Expression of Cecropin A and Lysozyme Heteropeptide in Pichia pastoris and Its Activity Analysis

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