黄鳝醛酮还原酶基因的重组表达及多抗制备
|
摘要
为实现原核表达黄鳝醛酮还原酶基因并制备其多克隆抗体,笔者利用基因特异性引物自黄鳝肝脏cDNA中扩增黄鳝醛酮还原酶全长序列,将之克隆至原核表达载体pET-28a(+)中,构建了重组表达载体;经测序验证后的重组质粒转化大肠杆菌BL21(DE3)后进行IPTG诱导。利用Ni离子亲和柱纯化了重组蛋白,通过多次免疫新西兰兔制备了多克隆抗体;采用Western blot和免疫组化法鉴定兔抗醛酮还原酶多克隆抗体的特异性,用间接ELISA技术检测多抗的效价。结果发现成功构建pET/Eakr原核表达载体,并实现了重组蛋白的融合表达和纯化;Western blot检测表明制备的兔多克隆抗体不仅能特异性地识别来源于黄鳝4种组织的醛酮还原酶蛋白而且还可识别来源于黄颡鱼、团头鲂、乌鳢、鲫鱼和草鱼的同源蛋白。免疫组化结果提示该多抗可特异性识别黄鳝小肠、肝胰组织和脾脏的醛酮还原酶蛋白。制备的多抗效价达1∶6400。
A pair of gene-specific primers were synthesized to isolate the sequence of AKR of swamp eel Monopterus albus from the liver cDNAs to recombinant expression of swamp eel aldo-keto reductase in Escherichia coli and to prepare polyclonal antibody against it.The purified products were subcloned into expression vector pET-28 a(+).Subsequently,the confirmed plasmid pET/Eakr was transformed into BL21(DE3)competent cells.The positive clones were selected and were induced by IPTG.Then,the recombinant protein was purified by one step affinity chromatography with a Ni-NTA agarose column.New Zealand white rabbits were immunized with the purified protein to generate polyclonal antibodies.Western-blotting and immunohistochemistry assay were performed to detect the specificity of the antibody.Indirect ELISA was used to examine the titer of the antibody.The results showed that the antiserum specifically recognized Eakr in four different tissues of swamp eel and reacted with homologous proteins in grass carp Ctenopharyngodon idella,yellow catfish Pelteobagrus fulvidraco,bluntnose black bream Megalobrama amblycephala,snakehead Channa argus and crucian carp Carassius auratus.Immunohistochemistry assay revealed that the antiserum specifically reacted to Eakr in kidney,liver and intestine with antibody titer of about 1∶6400.
A pair of gene-specific primers were synthesized to isolate the sequence of AKR of swamp eel Monopterus albus from the liver cDNAs to recombinant expression of swamp eel aldo-keto reductase in Escherichia coli and to prepare polyclonal antibody against it.The purified products were subcloned into expression vector pET-28 a(+).Subsequently,the confirmed plasmid pET/Eakr was transformed into BL21(DE3)competent cells.The positive clones were selected and were induced by IPTG.Then,the recombinant protein was purified by one step affinity chromatography with a Ni-NTA agarose column.New Zealand white rabbits were immunized with the purified protein to generate polyclonal antibodies.Western-blotting and immunohistochemistry assay were performed to detect the specificity of the antibody.Indirect ELISA was used to examine the titer of the antibody.The results showed that the antiserum specifically recognized Eakr in four different tissues of swamp eel and reacted with homologous proteins in grass carp Ctenopharyngodon idella,yellow catfish Pelteobagrus fulvidraco,bluntnose black bream Megalobrama amblycephala,snakehead Channa argus and crucian carp Carassius auratus.Immunohistochemistry assay revealed that the antiserum specifically reacted to Eakr in kidney,liver and intestine with antibody titer of about 1∶6400.
引文
[1]Jez J M,Bennett M J,Schelgel B P,et al.Comparative anatomy of the aldo-keto reductase superfamily[J].Biochem Journal,1997,326(3):625-636.
[2]Penning T M.The aldo-keto reductases(AKRs):overview[J].Chemico-Biological Interactions,2015(234):236-246.
[3]Mindnich R D,Penning T M.Aldo-keto reductase(AKR)superfamily:genomics and annotation[J].Human Genomics,2009,3(4):362-370.
[4]Sanli G,Dudley J I,Blaber M.Structural biology of the aldo-keto reductase family of enzymes[J].Cell Biochemistry and Biophysics,2003,3(1):79-101.
[5]Sengupta D,Naik D,Reddy A R.Plant aldo-keto reductases(AKRs)as multi-tasking soldiers involved in diverse plant metabolic processes and stress defense:a structure-function update[J].Journal of Plant Physiology,2015(179):40-55.
[6]Kanayama Y,Mizutani R,Yaguchi S,et al.Characterization of an uncharacterized aldo-keto reductase gene from peach and its role in abiotic stress tolerance[J].Phytochemistry,2014,104(3):30-36.
[7]Chang Q,Harter T M,Rikimaru L T,et al.Aldo/keto reductases as modulators of stress response[J].Chemico-Biological Interactions,2003,143/144(2):325-332.
[8]Ri6ner T L,Penning T M.Role of aldo-keto reductase family 1(AKR1)enzymes in human steroid metabolism[J].Steroids,2014,79(1):49-63.
[9]Matsumoto R,Tsuda M,Yoshida K,et al.Aldo-keto reductase 1C1induced by interleukin-1βmediates the invasive potential and drug resistance of metastatic bladder cancer cells[J].Scientific Reports,2016(6):34625.
[10]Chow R K,Sin S T,Liu M,et al.AKR7A3suppresses tumorigenicity and chemoresistance in hepatocellular carcinoma through attenuation of ERK,c-Jun and NF-κB signaling pathways[J].Oncotarget,2016,doi:10.18632/oncotarget.12726.
[11]Davydov V V.Age-specific peculiarities of modulation of blood aldo-keto reductase isoenzyme spectrum[J].Bulletin of Experimental Biology and Medicine,2015,160(2):199-201.
[12]Endo S,Matsunaga T,Mamiya H,et al.Characterization of a rat NADPH-dependent aldoketo reductase(AKR1B13)induced by oxidative stress[J].ChemicoBiological Interactions,2009,178(1/3):151-157.
[13]李丹,张岐山,周立娜,等.敲减AKR1A1基因对H2O2及4-羟基壬烯醛诱导的1321N1脑星形细胞瘤细胞损伤的影响[J].细胞与分子免疫学杂志,2013,29(3):273-276.
[14]张秋艳,龚晓健.多元醇通路:糖尿病性白内障药物干预的重要靶标[J].药学进展,2013,37(4):145-152.
[15]蔡秋香,廖端芳,祖旭宇,等.醛酮还原酶超家族的研究进展[J].医学综述,2008,14(24):3693-3695.
[16]Osorio-Yá1ez C,García-Tavera J L,Pérez-Nú1ez M T,et al.Benzo(a)pyrene induces hepatic AKR1A1mRNA expression in tilapia fish(Oreochromis niloticus)[J].Toxicology Mechanisms and Methods,2012,22(6):438-444.
[17]Hyndman D,Bauman D R,Heredia V V,et al.The aldo-keto reductase superfamily homepage[J].ChemicoBiological Interactions,2003,143/144(2):621-631.
[18]Li D,Ellis E M,Ferrari M.Human aldo-keto reductase AKR7A2protects against the cytotoxicity and mutagenicity of reactive aldehydes and lowers intracellular reactive oxygen species in hamster V79-4 cells[J].Chemico-Biological Interactions,2012,195(1):25-34.
[19]Zhang L,Zhang H,Zhao Y.Inhibitor selectivity between aldo-keto reductase superfamily members AKR1B10and AKR1B1:role of Trp112(Trp111)[J].FEBS Letters,2013,587(22):3681-3686.
[20]Huang L,He R,Luo W,et al.Aldo-keto reductase family 1 member B10inhibitors:potential drugs for cancer treatment.[J].Recent Pat Anticancer Drug Discov,2016,11(2):184-196.
[21]Lapthorn A J,Zhu X,Ellis E M.The diversity of microbial aldo/keto reductases fromEscherichia coli K12[J].Chemico-Biological Interactions,2013,202(1/3):168-177.
[22]Macleod A K,Kelly V P,Higgins L G,et al.Expression and localization of rat aldo-keto reductases and induction of the 1B13and 1D2isoforms by phenolic antioxidants[J].Drug Metabolism&Disposition the Biological Fate of Chemicals,2010,38(2):341-346.
[23]崔亚飞,姚学萍,雍康,等.奶牛S100A12蛋白多克隆抗血清的制备[J].动物医学进展,2012,33(6):77-80.
[2]Penning T M.The aldo-keto reductases(AKRs):overview[J].Chemico-Biological Interactions,2015(234):236-246.
[3]Mindnich R D,Penning T M.Aldo-keto reductase(AKR)superfamily:genomics and annotation[J].Human Genomics,2009,3(4):362-370.
[4]Sanli G,Dudley J I,Blaber M.Structural biology of the aldo-keto reductase family of enzymes[J].Cell Biochemistry and Biophysics,2003,3(1):79-101.
[5]Sengupta D,Naik D,Reddy A R.Plant aldo-keto reductases(AKRs)as multi-tasking soldiers involved in diverse plant metabolic processes and stress defense:a structure-function update[J].Journal of Plant Physiology,2015(179):40-55.
[6]Kanayama Y,Mizutani R,Yaguchi S,et al.Characterization of an uncharacterized aldo-keto reductase gene from peach and its role in abiotic stress tolerance[J].Phytochemistry,2014,104(3):30-36.
[7]Chang Q,Harter T M,Rikimaru L T,et al.Aldo/keto reductases as modulators of stress response[J].Chemico-Biological Interactions,2003,143/144(2):325-332.
[8]Ri6ner T L,Penning T M.Role of aldo-keto reductase family 1(AKR1)enzymes in human steroid metabolism[J].Steroids,2014,79(1):49-63.
[9]Matsumoto R,Tsuda M,Yoshida K,et al.Aldo-keto reductase 1C1induced by interleukin-1βmediates the invasive potential and drug resistance of metastatic bladder cancer cells[J].Scientific Reports,2016(6):34625.
[10]Chow R K,Sin S T,Liu M,et al.AKR7A3suppresses tumorigenicity and chemoresistance in hepatocellular carcinoma through attenuation of ERK,c-Jun and NF-κB signaling pathways[J].Oncotarget,2016,doi:10.18632/oncotarget.12726.
[11]Davydov V V.Age-specific peculiarities of modulation of blood aldo-keto reductase isoenzyme spectrum[J].Bulletin of Experimental Biology and Medicine,2015,160(2):199-201.
[12]Endo S,Matsunaga T,Mamiya H,et al.Characterization of a rat NADPH-dependent aldoketo reductase(AKR1B13)induced by oxidative stress[J].ChemicoBiological Interactions,2009,178(1/3):151-157.
[13]李丹,张岐山,周立娜,等.敲减AKR1A1基因对H2O2及4-羟基壬烯醛诱导的1321N1脑星形细胞瘤细胞损伤的影响[J].细胞与分子免疫学杂志,2013,29(3):273-276.
[14]张秋艳,龚晓健.多元醇通路:糖尿病性白内障药物干预的重要靶标[J].药学进展,2013,37(4):145-152.
[15]蔡秋香,廖端芳,祖旭宇,等.醛酮还原酶超家族的研究进展[J].医学综述,2008,14(24):3693-3695.
[16]Osorio-Yá1ez C,García-Tavera J L,Pérez-Nú1ez M T,et al.Benzo(a)pyrene induces hepatic AKR1A1mRNA expression in tilapia fish(Oreochromis niloticus)[J].Toxicology Mechanisms and Methods,2012,22(6):438-444.
[17]Hyndman D,Bauman D R,Heredia V V,et al.The aldo-keto reductase superfamily homepage[J].ChemicoBiological Interactions,2003,143/144(2):621-631.
[18]Li D,Ellis E M,Ferrari M.Human aldo-keto reductase AKR7A2protects against the cytotoxicity and mutagenicity of reactive aldehydes and lowers intracellular reactive oxygen species in hamster V79-4 cells[J].Chemico-Biological Interactions,2012,195(1):25-34.
[19]Zhang L,Zhang H,Zhao Y.Inhibitor selectivity between aldo-keto reductase superfamily members AKR1B10and AKR1B1:role of Trp112(Trp111)[J].FEBS Letters,2013,587(22):3681-3686.
[20]Huang L,He R,Luo W,et al.Aldo-keto reductase family 1 member B10inhibitors:potential drugs for cancer treatment.[J].Recent Pat Anticancer Drug Discov,2016,11(2):184-196.
[21]Lapthorn A J,Zhu X,Ellis E M.The diversity of microbial aldo/keto reductases fromEscherichia coli K12[J].Chemico-Biological Interactions,2013,202(1/3):168-177.
[22]Macleod A K,Kelly V P,Higgins L G,et al.Expression and localization of rat aldo-keto reductases and induction of the 1B13and 1D2isoforms by phenolic antioxidants[J].Drug Metabolism&Disposition the Biological Fate of Chemicals,2010,38(2):341-346.
[23]崔亚飞,姚学萍,雍康,等.奶牛S100A12蛋白多克隆抗血清的制备[J].动物医学进展,2012,33(6):77-80.