Improvement of Cecropin AD and Buforin Ⅱ Expression Vector and Optimization for Fusion Protein Expression Conditions
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摘要: 为提高重组大肠杆菌表达天蚕素AD和BuforinⅡ融合蛋白的表达效率, 研究表达菌株、质粒稳定性和诱导条件对天蚕素AD和BuforinⅡ融合蛋白表达的影响。通过SDS-PAGE分析目的蛋白占全菌总蛋白的含量, 确定大肠杆菌BER2566为表达菌株;利用基因工程技术, 构建卡那霉素抗性的表达质粒pET (K) -Trx-CADBuforinII, 提高质粒稳定性和蛋白表达效率;优化了IPTG浓度、诱导时机和诱导时间等诱导条件, 确定菌体浓度为OD600=0.8时, IPTG浓度为0.8mmol·L-1, 诱导时间为5h, 目的蛋白表达量最高, 可达到全菌总蛋白含量的50%以上。Abstract: Effects of expression strains, plasmid stability and inducing conditions were studied on improving the expression efficiency of Cecropin AD and Buforin Ⅱ fusion protein in recombinant Escherichia coli.According to the result of SDS-PAGE, E.coli BER2566was accepted as expression strain.Plasmid pET (K) -Trx-CAD-Buforin Ⅱ with kanamycin resistant gene was constructed to improve the plasmid stability and protein expression.Inducing conditions including IPTG concentration, induction time and inducing time length were optimized.With the optimal conditions, the highest expression level of fusion protein was obtained in recombinant E.coli at the level of OD600 0.8and final IPTG concentration of 0.8mmol·L-1 for 5hinducing, which was more than 50%of the total protein in E.coli.
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Key words:
- E.coli /
- cecropin AD /
- buforin Ⅱ /
- protein expression
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[1] GUAN-GUERRA E, SANTOS-MENDOZA T, LUGO-REYES S O, et al.Antimicrobial peptides:general overview and clinical implications in human health and disease[J].Clinical Immunology, 2010, 135 (1) :1-11. [2] 郭伟生, 吴哲, 郭海勇, 等.抗菌肽的基因克隆表达及其在畜牧业中的应用前景[J].吉林畜牧兽医, 2008, 29 (12) :10-12. [3] PAPAGIANNI M.Ribosomally synthesized peptides with antimicrobial properties:biosynthesis, structure, function, and applications[J].Biotechnology advances, 2003, 21 (6) :465-499. [4] HANCOCK R E.Cationic peptides:effectors in innate immunity and novel antimicrobials[J].The Lancet infectious diseases, 2001, 1 (3) :156-164. [5] 郑青, 苏智慧.抗菌肽AD基因的合成[J].生物化学与生物物理进展, 1998, 25 (2) :178-180. [6] KIM S, PARK C-B, LEE D-K, et al.A Novel Antimicrobial Peptide Isolated From Bufo Bufo Gargarizans[P].WO Patent, 1998, 007, 440. [7] GIACOMETTI A, CIRIONI O, GHISELLI R, et al.Potential therapeutic role of cationic peptides in three experimental models of septic shock[J].Antimicrobial agents and chemotherapy, 2002, 46 (7) :2132-2136. [8] GHISELLI R, GIACOMETTI A, CIRIONI O, et al.Cationic peptides combined with betalactams reduce mortality from peritonitis in experimental rat model[J].Journal of Surgical Research, 2002, 108 (1) :107-111. [9] 扈进冬, 魏艳丽, 张广志, 等.天蚕素AD和蛙BuforinⅡ融合蛋白在大肠杆菌中的表达[J].中国畜牧兽医, 2012, 39 (4) :35-38. [10] ANDREU D, UBACH J, BOMAN A, et al.Shortened cecropin A-melittin hybrids significant size reduction retains potent antibiotic activity[J].FEBS letters, 1992, 296 (2) :190-194. [11] LI Y.Recombinant production of antimicrobial peptides in Escherichia coli:A review[J].Protein expression and purification, 2011, 80 (2) :260-267. [12] 赵东红, 戴祝英, 周开亚.昆虫抗菌肽的功能, 作用机理与分子生物学研究最新进展[J].生物工程进展, 1999, 19 (5) :14-17. [13] 陆海荣, 李国栋, 吴宏宇, 等.抗菌肽GK1在大肠杆菌中的融合表达[J].生物工程学报, 2008, 24 (1) :21-26. [14] ZHOU L, ZHAO Z, LI B, et al.TrxA mediating fusion expression of antimicrobial peptide CM4from multiple joined genes in Escherichia coli[J].Protein expression and purification, 2009, 64 (2) :225-230. [15] 王继雯, 谢宝恩, 周伏忠, 等.抗菌肽hepcidin融合蛋白在大肠杆菌中表达条件的优化[J].生物技术通报, 2009, 10:151-155. [16] 阿里木热·汗古丽, 毛新芳, 刘忠渊.黄粉虫抗菌肽在大肠杆菌中表达条件优化及活性分析[J].生物工程学报, 2013, (6) :836-847.
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