利用小泛素蛋白修饰分子融合技术在大肠杆菌BL21(DE3)中表达重组抗菌肽LLv  

Expression of Recombinant Antimicrobial Peptide LLv in E.coli BL21(DE3)Using SUMO Fusion Technology

作　　者：殷文霞 王为栋[1] 刘晓东[1] 邵坤 单虎[1] 张洪亮[1] 王述柏[2] YIN Wenxia;WANG Weidong;LIU Xiaodong;SHAO Kun;SHAN Hu;ZHANG Hongliang;WANG Shubai(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109 [2]青岛农业大学动物科技学院,山东青岛266109

出　　处：《青岛农业大学学报（自然科学版）》2023年第4期265-269,共5页Journal of Qingdao Agricultural University(Natural Science)

基　　金：青岛市动物保健品行业智库联合基金二期科技服务计划(18-6-3-1-jch);山东省农业重大应用技术创新课题;国际合作项目(2014DFR30980)。

摘　　要：为了研究在大肠杆菌(E.coli)BL21(DE3)中利用小分子泛素蛋白(small ubiquitin-like modifier,SUMO)融合技术表达重组抗菌肽LLv(antimicrobial peptide LLv)的方法。试验以天然抗菌肽LL-37氨基酸组成及分子结构为依据,人工设计了LLv的氨基酸序列和基因序列。利用SUMO融合技术,构建重组大肠杆菌表达载体pSUMO-LLv在E.coli BL21(DE3)中表达SUMO-LLv,并研究异丙基硫代半乳糖苷(isopropyl beta-D-thiogalacyranoside,IPTG)浓度和诱导时间对融合蛋白SUMO-LLv表达的影响,同时分析融合蛋白SUMO-LLv表达的可溶性并分离纯化目的蛋白LLv。结果表明:建立的E.coli BL21(DE3)的pSUMO-LLv重组表达载体,经DNA测序验证构建正确;SUMO-LLv的诱导表达最佳条件是IPTG浓度0.5 mmol/L,诱导表达时间为2 h;融合蛋白SUMO-LLv主要存在于菌液上清液中,表达含量为11.34μg/mL;免疫印迹试验(Western blot)鉴定证明融合蛋白具有良好的反应原性;采用Ni柱亲和层析法在诱导菌液中成功纯化到了目的蛋白LLv,大小为5 kDa。说明在E.coli BL21(DE3)中利用SUMO融合技术表达重组抗菌肽LLv的方法可行。To investigate the expression of antimicrobial peptide LLv in E.coli BL21(DE3)by small ubiquitin-like modifier(SUMO)fusion technique.Based on the amino acid composition and molecular structure of natural antimicrobial peptide LL-37,the amino acid sequence and gene sequence of LLv were artificially designed.Using SUMO fusion technology,recombinant Escherichia coli(E.coli)expression vector pSUCO-LLv was constructed to express SUMO-LLv in E.coli BL21(DE3),and isopropyl beta-D-thiogalacyranoside,Isopropyl beta-d-Thiogalacyranoside was studied.The effect of IPTG concentration and induction time on the expression of fusion protein SUMO-LLv,and the solubility of fusion protein SUMO-LLv expression were analyzed and the target protein LLv was isolated and purified.The results showed that the pSUMO-LLv recombinant expression vector of E.coli BL21(DE3)was correctly constructed by DNA sequencing.The optimal conditions for inducing the expression of SUMO-LLv were IPTG concentration of 0.5 mmol/L and induction time of 2 h.The fusion protein SUMO-LLv mainly existed in the supernatant of bacterial solution,and the expression content was 11.34μg/mL.Western blot assay proved that the fusion protein had good reactivity.The target protein LLv with the size of 5 kDa was successfully purified from the induced bacterial solution by Ni column affinity chromatography.These results indicate that the SUMO fusion technique is a feasible method to express recombinant antimicrobial peptide LLv in E.coli BL21(DE3).

关 键 词：LLv SUMO 融合蛋白 诱导表达 E.coli BL21(DE3) 

分 类 号：S859.797[农业科学—临床兽医学]