磷脂酶A1辅助蛋白N端截短菌株的构建及其优化表达

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  • 1(安徽工程大学 生物与化学工程学院,安徽 芜湖,241000)2(微生物发酵安徽省工程研究中心,安徽 芜湖,241000)

网络出版日期: 2018-07-04

基金资助

国家自然科学基金(31471615);安徽工程大学研究生实践与创新项目(Y040116009)

Construction and optimization of the N-terminal truncated strain ofphospholipase A1 accessory protein

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  • 1(College of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu 241000, China) 2(Anhui Engineering Technology Research Center of Microbial Fermentation, Wuhu 241000, China)

Online published: 2018-07-04

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摘要

根据GenBank公布的磷脂酶A1辅助蛋白plaS基因序列,通过生物信息学软件分析,截短辅助蛋白PlaS的N端35AA。将截短后的辅助蛋白plaS基因经PCR扩增技术与原核表达载体pET-28a(+)连接,再转化到BL21(DE3)宿主菌中进行融合表达,从而成功构建dSP28表达菌株。用异丙基-β-D-硫代半乳糖苷(isopropy-β-D-thiogalactoside, IPTG)诱导辅助蛋白表达,对诱导时间、IPTG浓度、起始菌体浓度(OD600nm)和温度逐项进行优化,摸索得到辅助蛋白的最佳诱导表达条件为诱导时间8 h,IPTG浓度0.2 mmol/L,起始体浓度(OD600nm)0.7,温度40 ℃,转速200 r/min。在此条件下,对P28、SP28和dSP28发酵中OD600nm进行测定,结果表明,加入IPTG诱导后P28和dSP28能够快速的增殖,且诱导8 h后仍显示增长趋势,而SP28的生长受到明显的抑制作用。

本文引用格式

朱昊, 薛正莲, 王洲, 等 . 磷脂酶A1辅助蛋白N端截短菌株的构建及其优化表达[J]. 食品与发酵工业, 2018 , 44(5) : 34 -38 . DOI: 10.13995/j.cnki.11-1802/ts.016533

Abstract

According to phospholipase A1 accessory protein sequences of plaS from GenBank, 35 amino acids at N-terminal of PlaS was removed based on bioinformatics analysis.The truncated accessory proteins plaS gene was amplified by PCR and inserted into prokaryotic expression vector pET-28a (+) connection, and then the plasmid was transformed into BL21 (DE3) host bacteria for fusion expression, thereby successfully constructed dSP28 expression strain.IPTG was used to induce the expression of accessory proteins.The induction time, IPTG concentration, initial bacterial concentration (OD600nm) and temperature were optimized step by step.The optimum induction conditions were as follows: the induction time was 8 hours, the IPTG concentration was 0.2 mmol/L, the initial concentration (OD600nm) was 0.7, the temperature was 40 ℃, the rotation speed was 200 r/min.Under these conditions, OD600nm was determined in P28, SP28 and dSP28 fermentations.The results showed that P28 and dSP28 could proliferate rapidly after induction with IPTG, and the growth of P28 and dSP28 could be increased even after induction for 8 h, and the growth of SP28 was inhibited obviously.
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