尼罗罗非鱼Galectin-3基因的原核表达与条件优化
|
摘要
【目的】对Galectin-3蛋白进行纯化,优化诱导相关表达条件,为大量获取罗非鱼Galectin-3蛋白提供方案。【方法】根据NCBI上已公布的罗非鱼(Oreochromismossambicus)Galectin-3基因序列,设计多对带Eco RI和Xhol酶切位点的引物,筛选出良性扩增引物,对罗非鱼cDNA经进行聚合酶链式反应(PCR)扩增、限制性快切酶双酶切、T4连接酶连接等步骤,构建罗非鱼Galectin-3基因的原核表达质粒pGEX-4T-Galectin-3,将重组质粒导入大肠杆菌BL21中,进行Galectin-3重组蛋白的表达。并比较不同的诱导温度、诱导时间、IPTG浓度条件下的表达效果,对Galectin-3蛋白表达最优条件进行筛选。【结果】构建了罗非鱼Galectin-3原核表达质粒,进行Galectin-3重组蛋白后发现37℃下,0.4 mmol/L浓度的IPTG诱导5 h即可诱导Galectin-3重组蛋白高效表达。免疫印迹结果显示,经蛋白纯化柱纯化后的pGEX-4T-Galectin-3重组蛋白可与GST-Tag单克隆抗体发生特异性反应,进而表明表达的重组蛋白是罗非鱼的Galectin-3蛋白。【结论】本研究成功构建了罗非鱼Galectin-3基因的原核表达载体,并确定了Galectin-3融合蛋白最适的表达条件:37℃下,0.4 mmol/L浓度的IPTG诱导5 h。
【Objective】The prokaryotic expression and expression conditions of galectin-3 gene were optimized to obtain a large amount of proteins, laying a foundation for further preparation of monoclonal antibodies. 【 Methods 】 Based on the published sequence of Galicon(Oreochromis mossambicus) Galectin-3 gene on NCBI, several pairs of primers with Eco RI and Xhol restriction sites were designed to screen for benign amplification primers, and the tilapia cDNA was polymerase-chained.The prokaryotic expression plasmid pGEX-4 T-Galectin-3 of tilapia Galectin-3 gene was constructed by reaction(PCR) amplification, restriction enzyme digestion and T4 ligase ligation, and the recombinant plasmid was introduced into E. coli BL21. In the expression of Galectin-3 recombinant protein, we compared the expression effects of different induction temperature, induction time and IPTG concentration, and screened the optimal conditions for Galectin-3 protein expression. 【Results】pGEX-4 T-Galectin-3 was successfully constructed and the Galectin-3 recombinant protein was expressed. It was found that the high expression of Galectin-3 recombinant protein was induced by IPTG at a concentration of 0.4 mmol/L for 5 h at 37 ℃. Western blot results showed that the recombinant protein pGEX-4 T-Galectin-3 purified by protein purification column could specifically react with GST-Tag monoclonal antibody, indicating that the expressed recombinant protein is Galectin-3 protein of tilapia. 【Conclusion】In this study, the prokaryotic expression vector of the tilapia Galectin-3 gene was successfully constructed, and the optimal expression conditions of the Galectin-3 fusion protein were determined. The highest concentration was obtained by inducing IPTG at a concentration of 0.4 mmol/L for 5 h at 37 ℃.
【Objective】The prokaryotic expression and expression conditions of galectin-3 gene were optimized to obtain a large amount of proteins, laying a foundation for further preparation of monoclonal antibodies. 【 Methods 】 Based on the published sequence of Galicon(Oreochromis mossambicus) Galectin-3 gene on NCBI, several pairs of primers with Eco RI and Xhol restriction sites were designed to screen for benign amplification primers, and the tilapia cDNA was polymerase-chained.The prokaryotic expression plasmid pGEX-4 T-Galectin-3 of tilapia Galectin-3 gene was constructed by reaction(PCR) amplification, restriction enzyme digestion and T4 ligase ligation, and the recombinant plasmid was introduced into E. coli BL21. In the expression of Galectin-3 recombinant protein, we compared the expression effects of different induction temperature, induction time and IPTG concentration, and screened the optimal conditions for Galectin-3 protein expression. 【Results】pGEX-4 T-Galectin-3 was successfully constructed and the Galectin-3 recombinant protein was expressed. It was found that the high expression of Galectin-3 recombinant protein was induced by IPTG at a concentration of 0.4 mmol/L for 5 h at 37 ℃. Western blot results showed that the recombinant protein pGEX-4 T-Galectin-3 purified by protein purification column could specifically react with GST-Tag monoclonal antibody, indicating that the expressed recombinant protein is Galectin-3 protein of tilapia. 【Conclusion】In this study, the prokaryotic expression vector of the tilapia Galectin-3 gene was successfully constructed, and the optimal expression conditions of the Galectin-3 fusion protein were determined. The highest concentration was obtained by inducing IPTG at a concentration of 0.4 mmol/L for 5 h at 37 ℃.
引文
[1]DIAS R D O,MACHADO L D S,LUDOVICO M,et al.Insights into animal and plant lectins with antimicrobial activities[J].Molecules.2015,20(1):519-541.
[2]ZHU J J,WEI M,WANG Q H,et al.Characterization and expression of galectin-3 after Streptococcus agalactiae and Aeromonas hydrophila challenge in GIFT strain Nile tilapia(Oreochromis niloticus)[J].Fish and Shellfish Immunology,2019,86:974-980
[3]夏焱.家蝇蛹凝集素的分离纯化及对鼠腹腔巨噬细胞免疫功能的影响[D].天津:天津科技大学,2012.
[4]ELSAYED A F M.Total replacement of fish meal with animal protein sources in Nile tilapia,Oreochromis niloticus(L),feeds[J].Aquaculture Research,1998,29(4):275-280.
[5]YIN X,MU L,BIAN X,et al.Expression and functional characterization of transferrin in Nile tilapia(Oreochromis niloticus)in response to bacterial infection[J].Fish Shellfish Immunol,2018,74:530-539.
[6]YANG R Y,HSU D K,LIU F T.Expression of galectin-3modulates T-cell growth and apoptosis[J].Proceedings of the National Academy of Sciences,1996,93(13):6737-6742.
[7]MACKINNON A,FARNWORTH S P,HENDERSON N,et al.Regulation of alternative macrophage activation by Galectin-3[J].Journal of Immunology,2008,180(4):2650-2658.
[8]许崇利,杨梅,许崇波.大肠杆菌表达系统的影响因素[J].中国动物检疫,2010,27(8):66-68.
[9]曹红,仇燕,王刚.外源基因在大肠杆菌中表达的优化[J].河北师范大学学报,2004,28(1):82-85.
[10]陆海,吴薇,曾庆银,等.大肠杆菌BL21(DE3)中表达重组蛋白的研究[J].北京林业大学学报,2001,23(6):1-4.
[11]吴一凡,张双全,高秀玉,等.乳糖诱导p ET载体表达重组蛋白的研究[J].南京师范大学学报(自然科学版),2002,25(1):89-93.
[12]杨丽丽.斑马鱼甘露糖结合凝集素基因的表达和免疫功能的研究[D].青岛:中国海洋大学,2014.
[13]卢晓,陈政良.半乳糖凝集素[J].免疫学杂志,2004,20(s1):21-23.
[14]PENNACCHI Y,LEEF M J,CROSBIE P B,et al.Evidence of immune and inflammatory processes in the gills of AGD-affected Atlantic salmon,Salmo salar L.[J].Fish&Shellfish Immunology,2014,36(2):563-570.
[15]XU Z,PARRA D,G?MEZ D,et al.Teleost skin,an ancient mucosal surface that elicits gut-like immune responses[J].Proceedings of the National Academy of Sciences of the United States of America.2013,110(32):13097-13102.
[16]陆志款.复方中草药对红笛鲷生长及免疫功能的影响[J].当代水产,2013(11):74-75.
[17]HEDRICK R P,MACCONNELL E,KINKELIN P D.Proliferative kidney disease of salmonid fish[J].Annual Review of Fish Diseases,1993,3(3):277-290.
[18]朱峰.家蚕微孢子虫孢壁蛋白SWP26及其宿主互作蛋白的研究[D].镇江:江苏科技大学,2013.
[19]SHARROCKS A D.A T7 expression vector for producing N-and C-terminal fusion proteins with glutathione S-transferase[J].Gene,1994,138(1/2):105-108.
[20]牛金中,黄瑜,汤菊芬,等.尼罗罗非鱼补体3基因片段的原核表达及条件优化[J].广东海洋大学学报,2018,38(2):80-84.
[21]余波,程安春,汪铭书.大肠杆菌中重组蛋白可溶性表达的研究进展及展望[J].黑龙江畜牧兽医,2008,50(10):19-21.
[22]VILLAVERDE A,M.MAR CARRIó.Protein aggregation in recombinant bacteria:biological role of inclusion bodies[J].Biotechnology Letters,2003,25(17):1385-1395.
[23]MARSTON F A.The purification of eukaryotic polypeptides synthesized in Escherichia coli[J].Biochemical Journal,1986,240(1):1-12.
[24]苏裕.大肠杆菌中重组蛋白可溶性表达的研究[D].广州:华东师范大学,2007.
[25]黄瑜,张雪利,鲁义善,等.红笛鲷tdt基因融合蛋白原核表达条件的优化及纯化[J].广东海洋大学学报,2013,33(1):44-49.
[26]潘滨,吴建祥,李桂新,等.烟草曲茎病毒复制相关蛋白基因原核表达条件优化[J].浙江大学学报(农业与生命科学版),2007,51(1):24-28.
[27]王秋霞,张美英,张改平,等.猪囊尾蚴排泄分泌抗原Ts881蛋白原核表达条件的优化[J].华北农学报,2009,24(3):59-63.
[28]史继静,刘朝奇,杨凡,等.人IL-6基因的克隆及其在原核生物中表达及条件的优化[J].生物技术通报,2010,58(5):135-140.
[2]ZHU J J,WEI M,WANG Q H,et al.Characterization and expression of galectin-3 after Streptococcus agalactiae and Aeromonas hydrophila challenge in GIFT strain Nile tilapia(Oreochromis niloticus)[J].Fish and Shellfish Immunology,2019,86:974-980
[3]夏焱.家蝇蛹凝集素的分离纯化及对鼠腹腔巨噬细胞免疫功能的影响[D].天津:天津科技大学,2012.
[4]ELSAYED A F M.Total replacement of fish meal with animal protein sources in Nile tilapia,Oreochromis niloticus(L),feeds[J].Aquaculture Research,1998,29(4):275-280.
[5]YIN X,MU L,BIAN X,et al.Expression and functional characterization of transferrin in Nile tilapia(Oreochromis niloticus)in response to bacterial infection[J].Fish Shellfish Immunol,2018,74:530-539.
[6]YANG R Y,HSU D K,LIU F T.Expression of galectin-3modulates T-cell growth and apoptosis[J].Proceedings of the National Academy of Sciences,1996,93(13):6737-6742.
[7]MACKINNON A,FARNWORTH S P,HENDERSON N,et al.Regulation of alternative macrophage activation by Galectin-3[J].Journal of Immunology,2008,180(4):2650-2658.
[8]许崇利,杨梅,许崇波.大肠杆菌表达系统的影响因素[J].中国动物检疫,2010,27(8):66-68.
[9]曹红,仇燕,王刚.外源基因在大肠杆菌中表达的优化[J].河北师范大学学报,2004,28(1):82-85.
[10]陆海,吴薇,曾庆银,等.大肠杆菌BL21(DE3)中表达重组蛋白的研究[J].北京林业大学学报,2001,23(6):1-4.
[11]吴一凡,张双全,高秀玉,等.乳糖诱导p ET载体表达重组蛋白的研究[J].南京师范大学学报(自然科学版),2002,25(1):89-93.
[12]杨丽丽.斑马鱼甘露糖结合凝集素基因的表达和免疫功能的研究[D].青岛:中国海洋大学,2014.
[13]卢晓,陈政良.半乳糖凝集素[J].免疫学杂志,2004,20(s1):21-23.
[14]PENNACCHI Y,LEEF M J,CROSBIE P B,et al.Evidence of immune and inflammatory processes in the gills of AGD-affected Atlantic salmon,Salmo salar L.[J].Fish&Shellfish Immunology,2014,36(2):563-570.
[15]XU Z,PARRA D,G?MEZ D,et al.Teleost skin,an ancient mucosal surface that elicits gut-like immune responses[J].Proceedings of the National Academy of Sciences of the United States of America.2013,110(32):13097-13102.
[16]陆志款.复方中草药对红笛鲷生长及免疫功能的影响[J].当代水产,2013(11):74-75.
[17]HEDRICK R P,MACCONNELL E,KINKELIN P D.Proliferative kidney disease of salmonid fish[J].Annual Review of Fish Diseases,1993,3(3):277-290.
[18]朱峰.家蚕微孢子虫孢壁蛋白SWP26及其宿主互作蛋白的研究[D].镇江:江苏科技大学,2013.
[19]SHARROCKS A D.A T7 expression vector for producing N-and C-terminal fusion proteins with glutathione S-transferase[J].Gene,1994,138(1/2):105-108.
[20]牛金中,黄瑜,汤菊芬,等.尼罗罗非鱼补体3基因片段的原核表达及条件优化[J].广东海洋大学学报,2018,38(2):80-84.
[21]余波,程安春,汪铭书.大肠杆菌中重组蛋白可溶性表达的研究进展及展望[J].黑龙江畜牧兽医,2008,50(10):19-21.
[22]VILLAVERDE A,M.MAR CARRIó.Protein aggregation in recombinant bacteria:biological role of inclusion bodies[J].Biotechnology Letters,2003,25(17):1385-1395.
[23]MARSTON F A.The purification of eukaryotic polypeptides synthesized in Escherichia coli[J].Biochemical Journal,1986,240(1):1-12.
[24]苏裕.大肠杆菌中重组蛋白可溶性表达的研究[D].广州:华东师范大学,2007.
[25]黄瑜,张雪利,鲁义善,等.红笛鲷tdt基因融合蛋白原核表达条件的优化及纯化[J].广东海洋大学学报,2013,33(1):44-49.
[26]潘滨,吴建祥,李桂新,等.烟草曲茎病毒复制相关蛋白基因原核表达条件优化[J].浙江大学学报(农业与生命科学版),2007,51(1):24-28.
[27]王秋霞,张美英,张改平,等.猪囊尾蚴排泄分泌抗原Ts881蛋白原核表达条件的优化[J].华北农学报,2009,24(3):59-63.
[28]史继静,刘朝奇,杨凡,等.人IL-6基因的克隆及其在原核生物中表达及条件的优化[J].生物技术通报,2010,58(5):135-140.