大菱鲆(Scophthalmus maximus)蛋白质二硫键异构酶SmPDIA3的表达分析和功能验证
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摘要
本研究利用SMART-RACE技术克隆了大菱鲆SmPDIA3基因。SmPDIA3基因cDNA序列全长2083bp,包括1479bp的开放阅读框,编码492个氨基酸, GenBank登录号:MG765516。组织表达分析发现:SmPDIA3基因在肠、鳃、肝脏等组织中均有表达,其中在肝脏中表达量最高,脑中最低。进一步研究了温度胁迫后SmPDIA3基因在肠和肝脏组织中的表达变化规律,发现随着胁迫温度的升高,SmPDIA3基因的相对表达量总体上升,并在28°C时达到最大。利用原核表达技术,构建了pET-28a-PDIA3原核表达载体,IPTG诱导后融合蛋白主要在上清中表达。将上清中的蛋白分离纯化后,利用Westernblot技术验证了目的蛋白的准确性。将纯化后的目的蛋白浓缩后,利用BCA蛋白浓度测定试剂盒测得蛋白浓度为243.18μg/mL。最后设计变性溶菌酶的复性实验,验证了SmPDIA3蛋白具有分子伴侣功能,能够辅助变性蛋白的正确折叠。
We cloned the SmPDIA3 gene of turbot Scophthalmus maximus using the SMART-RACE technique. The cDNA sequence of SmPDIA3 is 2083 bp in length and includes a 1479 bp open reading frame encoding a 492 amino acid signal peptide containing 17 amino acids. The predicted molecular weight is 54.92 kDa, the theoretical isoelectric point is5.40, and GenBank accession number is MG765516. Tissue expression analysis showed that SmPDIA3 gene was expressed in intestinal, iliac, liver, and other tissues, with the highest expression in the liver and the lowest in the brain. In addition,we studied the expression of SmPDIA3 gene in the intestine and liver tissues after a thermal stress. We found that the relative expression of SmPDIA3 gene increased with the increase of temperature, and reached the maximum at 28°C. Using prokaryotic expression technology, the pET-28 a-PDIA3 prokaryotic expression vector was constructed and the target protein was successfully expressed in the supernatant. Finally, the refolding experiment of denatured lysozyme was designed to verify the function of the SmPDIA3 protein. We found that SmPDIA3 protein can assist the correct folding of denatured proteins, and has obvious molecular chaperone function.
We cloned the SmPDIA3 gene of turbot Scophthalmus maximus using the SMART-RACE technique. The cDNA sequence of SmPDIA3 is 2083 bp in length and includes a 1479 bp open reading frame encoding a 492 amino acid signal peptide containing 17 amino acids. The predicted molecular weight is 54.92 kDa, the theoretical isoelectric point is5.40, and GenBank accession number is MG765516. Tissue expression analysis showed that SmPDIA3 gene was expressed in intestinal, iliac, liver, and other tissues, with the highest expression in the liver and the lowest in the brain. In addition,we studied the expression of SmPDIA3 gene in the intestine and liver tissues after a thermal stress. We found that the relative expression of SmPDIA3 gene increased with the increase of temperature, and reached the maximum at 28°C. Using prokaryotic expression technology, the pET-28 a-PDIA3 prokaryotic expression vector was constructed and the target protein was successfully expressed in the supernatant. Finally, the refolding experiment of denatured lysozyme was designed to verify the function of the SmPDIA3 protein. We found that SmPDIA3 protein can assist the correct folding of denatured proteins, and has obvious molecular chaperone function.
引文
马丽娟,吕宾,孟立娜等,2011.PDIA3在腹泻型肠易激综合征患者结肠黏膜中的蛋白表达及其意义.胃肠病学,16(4):218-221
王婷,黄智慧,马爱军等,2014.基于转录组数据的大菱鲆(Scophthalmus maximus)SNP标记开发及多态性分析.海洋与湖沼,45(6):1300-1307
王璐,2012.蛋白二硫键异构酶ERp57调控血小板活化和血栓形成的作用及其机制研究.苏州:苏州大学硕士学位论文
吕利霞,汤睿智,邢万金,2010.PDIA3的结构与功能及其在精卵融合过程中的作用.生物技术通报,(3):31-34,49
刘群,段明明,苑纯秀等,2015.日本血吸虫PDIA3的原核表达和多抗血清制备.中国动物传染病学报,23(1):33-38
刘慧萍,陈苏民,1998.蛋白质二硫键异构酶结构域的克隆与活性研究.见:材料科学与工程技术论文集.北京:中国科学技术协会
李勇,孙国祥,柳阳等,2011.温度对高密度循环海水养殖大菱鲆摄食、生长及消化酶的影响.渔业科学进展,32(6):17-24
余秀娟,赵丽艳,张晓光,2013.体外蛋白质复性方法及小分子添加剂在复性过程中的作用.河北北方学院学报(自然科学版),29(2):9-15
胡玥,陶丽媛,吕宾,2015.蛋白质二硫键异构酶A3--疾病治疗的新靶点.中国病理生理杂志,31(6):1145-1149
贾长虹,2007.人工伴侣与小分子添加剂协同辅助变性溶菌酶复性的研究.天津:天津大学硕士学位论文高淳仁,王印庚,马爱军等,2006.温度对大菱鲆幼鱼生长、成活率和体内蛋白酶活性的影响.海洋水产研究,27(6):33-36
郭建丽,田岳强,马爱军等,2015.大菱鲆(Scophthalmus maximus)抗鳗弧菌(Vibrio anguillarum)性状的微卫星分子标记研究.海洋与湖沼,46(1):157-164
Aureli C,Gaucci E,Arcangeli V et al,2013.ERp57/PDIA3 binds specific DNA fragments in a melanoma cell line.Gene,524(2):390-395
Chen H B,Huang H Q,2011.Proteomic analysis of methyl parathion-responsive proteins in Sparus latus liver.Fish&Shellfish Immunology,30(3):800-806
Ellerman D A,Myles D G,Primakoff P,2006.A role for sperm surface protein disulfide isomerase activity in gamete fusion:evidence for the participation of ERp57.Developmental Cell,10(6):831-837
Freedman R B,Hirst T R,Tuite M F,1994.Protein disulphide isomerase:building bridges in protein folding.Trends in Biochemical Sciences,19(8):331-336
Guo G G,Patel K,Kumar V et al,2002.Association of the chaperoneglucose-regulatedprotein58(GRP58/ER-60/ERp57)with Stat3 in cytosol and plasma membrane complexes.Journal of Interferon&Cytokine Research,22(5):555-563
Huang T S,Olsvik P A,Kr?vel A et al,2009.Stress-induced expression of protein disulfide isomerase associated 3(PDIA3)in Atlantic salmon(Salmo salar L.).Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,154(4):435-442
Ndong D,Chen Y Y,Lin Y H et al,2007.The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures.Fish&Shellfish Immunology,22(6):686-694
Pendyala G,Ninemire C,Fox H S,2012.Protective role for the disulfide isomerase PDIA3 in methamphetamine neurotoxicity.PLoS One,7(6):e38909
Sever L,Bols N C,Dixon B,2013.The cloning and inducible expression of the rainbow trout ERp57 gene.Fish&Shellfish Immunology,34(2):410-419
Turano C,Coppari S,Altieri F et al,2002.Proteins of the PDIfamily:unpredicted non-ER locations and functions.Journal of Cellular Physiology,193(2):154-163
Zhang X Q,Pan Y,Yu C H et al,2015.PDIA3 knockdown exacerbates free fatty acid-induced hepatocyte steatosis and apoptosis.PLoS One,10(7):e0133882
Zhuang Z M,Wang X T,Zhang L et al,2015.The effect of PDIA3 gene knockout on the mucosal immune function in IBS rats.International Journal of Clinical and Experimental Medicine,8(5):6866-6877
王婷,黄智慧,马爱军等,2014.基于转录组数据的大菱鲆(Scophthalmus maximus)SNP标记开发及多态性分析.海洋与湖沼,45(6):1300-1307
王璐,2012.蛋白二硫键异构酶ERp57调控血小板活化和血栓形成的作用及其机制研究.苏州:苏州大学硕士学位论文
吕利霞,汤睿智,邢万金,2010.PDIA3的结构与功能及其在精卵融合过程中的作用.生物技术通报,(3):31-34,49
刘群,段明明,苑纯秀等,2015.日本血吸虫PDIA3的原核表达和多抗血清制备.中国动物传染病学报,23(1):33-38
刘慧萍,陈苏民,1998.蛋白质二硫键异构酶结构域的克隆与活性研究.见:材料科学与工程技术论文集.北京:中国科学技术协会
李勇,孙国祥,柳阳等,2011.温度对高密度循环海水养殖大菱鲆摄食、生长及消化酶的影响.渔业科学进展,32(6):17-24
余秀娟,赵丽艳,张晓光,2013.体外蛋白质复性方法及小分子添加剂在复性过程中的作用.河北北方学院学报(自然科学版),29(2):9-15
胡玥,陶丽媛,吕宾,2015.蛋白质二硫键异构酶A3--疾病治疗的新靶点.中国病理生理杂志,31(6):1145-1149
贾长虹,2007.人工伴侣与小分子添加剂协同辅助变性溶菌酶复性的研究.天津:天津大学硕士学位论文高淳仁,王印庚,马爱军等,2006.温度对大菱鲆幼鱼生长、成活率和体内蛋白酶活性的影响.海洋水产研究,27(6):33-36
郭建丽,田岳强,马爱军等,2015.大菱鲆(Scophthalmus maximus)抗鳗弧菌(Vibrio anguillarum)性状的微卫星分子标记研究.海洋与湖沼,46(1):157-164
Aureli C,Gaucci E,Arcangeli V et al,2013.ERp57/PDIA3 binds specific DNA fragments in a melanoma cell line.Gene,524(2):390-395
Chen H B,Huang H Q,2011.Proteomic analysis of methyl parathion-responsive proteins in Sparus latus liver.Fish&Shellfish Immunology,30(3):800-806
Ellerman D A,Myles D G,Primakoff P,2006.A role for sperm surface protein disulfide isomerase activity in gamete fusion:evidence for the participation of ERp57.Developmental Cell,10(6):831-837
Freedman R B,Hirst T R,Tuite M F,1994.Protein disulphide isomerase:building bridges in protein folding.Trends in Biochemical Sciences,19(8):331-336
Guo G G,Patel K,Kumar V et al,2002.Association of the chaperoneglucose-regulatedprotein58(GRP58/ER-60/ERp57)with Stat3 in cytosol and plasma membrane complexes.Journal of Interferon&Cytokine Research,22(5):555-563
Huang T S,Olsvik P A,Kr?vel A et al,2009.Stress-induced expression of protein disulfide isomerase associated 3(PDIA3)in Atlantic salmon(Salmo salar L.).Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,154(4):435-442
Ndong D,Chen Y Y,Lin Y H et al,2007.The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures.Fish&Shellfish Immunology,22(6):686-694
Pendyala G,Ninemire C,Fox H S,2012.Protective role for the disulfide isomerase PDIA3 in methamphetamine neurotoxicity.PLoS One,7(6):e38909
Sever L,Bols N C,Dixon B,2013.The cloning and inducible expression of the rainbow trout ERp57 gene.Fish&Shellfish Immunology,34(2):410-419
Turano C,Coppari S,Altieri F et al,2002.Proteins of the PDIfamily:unpredicted non-ER locations and functions.Journal of Cellular Physiology,193(2):154-163
Zhang X Q,Pan Y,Yu C H et al,2015.PDIA3 knockdown exacerbates free fatty acid-induced hepatocyte steatosis and apoptosis.PLoS One,10(7):e0133882
Zhuang Z M,Wang X T,Zhang L et al,2015.The effect of PDIA3 gene knockout on the mucosal immune function in IBS rats.International Journal of Clinical and Experimental Medicine,8(5):6866-6877