PROKARYOTIC EXPRESSION, ANTIBODY PREPARATION, AND REGULATION BY DIETARY STARCH LEVELS OF G6PC AND GCK IN MICROPTERUS SALMOIDES
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摘要:
为深入研究营养因子调控大口黑鲈(Micropterus salmoides)葡萄糖稳态的分子机制, 研究成功克隆并构建了大口黑鲈葡萄糖-6-磷酸酶催化亚基(G6pc)和葡萄糖激酶(Gck)截短序列的重组质粒载体, 转化至Escherichia coli Rosetta感受态细胞并以1 mmol/L IPTG (异丙基-β-D-硫代半乳糖苷)、16℃诱导表达过夜, 在细胞破碎后的上清中得到了大量表达。通过GST-tag亲和层析的方法纯化得到高纯度的G6pc和Gck截短重组蛋白后, 将其分别与弗氏佐剂乳化成免疫原, 免疫KM小鼠5次, 成功制备具有高特异性和高效价的多克隆抗体(G6pc和Gck抗体的效价分别大于1﹕3000和1﹕10000)。Westen blot检测发现大口黑鲈G6pc和Gck蛋白主要分布在肝脏中, 免疫荧光染色显示G6pc的阳性信号定位于细胞核周围, 而Gck的阳性信号散布整个细胞。在8周养殖实验结束后, 随饲料淀粉水平以6%的梯度从8%增加到20%, 大口黑鲈肝脏中糖酵解和糖原合成关键酶编码基因的表达及Gck水平逐渐升高, 但糖异生和糖原分解关键酶编码基因的表达及G6pc水平仅在14%淀粉水平时被抑制, 意味着在高淀粉水平(20%)下大口黑鲈肝脏中葡萄糖-G6P间的相互转化发生了无效循环。综上所述, 研究成功制备了大口黑鲈G6pc和Gck特异性识别的多克隆抗体, 探明了饲料淀粉水平对其表达的影响, 为深入研究G6pc和Gck在大口黑鲈葡萄糖稳态维持过程中的重要作用奠定了基础。
Abstract:The dysregulation of glucose-G6P (glucose-6-phosphate) interconversion is thought to be an important reason for the low glucose tolerance of carnivorous fish. However, it remains unclear if this phenomenon applies to largemouth bass (Micropterus salmoides, LMB). To investigate the regulatory mechanism of glucose homeostasis influenced by nutritional factors in LMB, we cloned and constructed recombinant plasmid vectors containing truncated sequences of glucose-6-phosphatase catalytic subunit (g6pc) and glucokinase (gck). These recombinant plasmid vectors were transformed into Escherichia coli Rosetta receptor cells and successfully expressed using 1 mmol/L IPTG (isopropyl-β-D-thiogalactopyranoside) induction overnight at 16℃. Following the lysis receptor cells, truncated G6pc, and Gck recombinant proteins were purified by GST-tag affinity chromatography from the supernatants. The purified recombinant proteins were emulsified with Freund's adjuvant to create immunogens, and immunized to KM mice for five times. G6pc and Gck polyclonal antibodies with high specificity were successfully prepared with titers exceeding 1﹕3000 and 1﹕10000, respectively. Western blot analysis showed that both G6pc and Gck in LMB were mainly distributed in the liver. Immunofluorescent staining indicated that the G6pc positive signals were localized around the nucleus, whereas Gck positive signals spread throughout the hepatocytes. After an 8-weeks feeding trial, the results showed that Gck level and the expression of genes involved in glycolysis and glycogenesis in the liver of LMB increased gradually with dietary starch levels rising from 8% to 20% in 6% increments. Conversely, G6pc levels and the expression of genes involved in gluconeogenesis and glycogenolysis were only down-regulated at 14% starch. These results suggested that a high starch diet (20%) could induce a futile cycle of glucose-G6P interconversion in the liver of LMB. In summary, specific polyclonal antibodies of G6pc and Gck were successfully prepared in LMB, and the regulation of their expression by dietary starch levels was evaluated in this study. This research lays the foundation for further elucidating the roles of G6pc and Gck in the glucose homeostasis of LMB.
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Keywords:
- G6Pase /
- Glucokinase /
- Prokaryotic expression /
- Polyclonal antibody /
- Micropterus salmoides
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图 1 大口黑鲈G6pc和Gck蛋白的理化性质和三级结构分析
A. G6pc; B. Gck; 截短后蛋白的氨基酸序列由黑色方框标出, 三级结构由深色表示
Figure 1. Physicochemical properties and structural analysis of deduced G6pc and Gck in largemouth bass
A. G6pc; B. Gck; Black boxes indicate amino acid sequences of truncated proteins; Tertiary structures are indicated by dark colors
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图 2 g6pc和gck基因的PCR扩增产物电泳分析
M. DNA分子量标准; 1. g6pc截短PCR产物; 2. gck截短PCR产物
Figure 2. Electrophoretic analysis of PCR products for g6pc and gck
M. DNA marker; 1. PCR product of truncated g6pc; 2. PCR product of truncated gck
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图 3 G6pc和Gck重组蛋白表达和纯化产物的SDS-PAGE分析
A. G6pc重组蛋白; B. Gck重组蛋白; M. 蛋白质分子量标准; 1. 未诱导空质粒; 2. 诱导后空质粒; 3. 未诱导截短重组质粒; 4. 诱导后截短重组质粒; 5. 破菌后沉淀; 6. 破菌后上清; 7. 纯化后重组蛋白
Figure 3. SDS-PAGE analysis of G6pc and Gck recombinant protein expression and purified products
A. recombinant G6pc; B. recombinant Gck. M. protein marker; 1. uninduced empty plasmid; 2: induced empty plasmid; 3. uninduced truncated recombinant plasmid; 4. induced truncated recombinant plasmid; 5. precipitation after bacterial fragmentation; 6. supernatant after bacterial fragmentation; 7. purified target recombinant protein
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图 4 Western blot鉴定G6pc和Gck重组蛋白
M. 蛋白质分子量标准; 1. GST标签蛋白; 2. G6pc重组蛋白; 3. Gck重组蛋白; 4. 肝组织蛋白
Figure 4. Identification of G6pc and Gck recombinant proteins through Western blot
M. protein marker; 1. GST-tag protein; 2. recombinant G6pc protein; 3. recombinant Gck protein; 4. liver protein
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图 5 G6pc和Gck多克隆抗体的特异性分析
A. G6pc多克隆抗体; B. Gck多克隆抗体; M. 蛋白质分子量标准; 1. GST标签蛋白; 2. 重组蛋白; 3. 肝组织蛋白
Figure 5. Specificity analysis of G6pc and Gck polyclonal antibodies
A. G6pc polyclonal antibody; B. Gck polyclonal antibody. M. protein marker; 1. GST-tag protein; 2. recombinant protein; 3. liver protein
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图 6 G6pc和Gck多克隆抗体的效价分析
稀释比例Dilution ratio: 1. 1﹕1000; 2. 1﹕3000; 3. 1﹕5000; 4. 1﹕10000; 5. 1﹕50000
Figure 6. Titer analysis of G6pc and Gck polyclonal antibodies
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图 7 大口黑鲈G6pc和Gck蛋白的组织分布
1. 肝脏liver; 2. 心脏heart; 3. 白肌white muscle; 4. 肠道intestine
Figure 7. Tissue distribution of G6pc and Gck in largemouth bass
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图 8 大口黑鲈肝组织G6pc和Gck蛋白的亚细胞定位
绿色代表G6pc阳性信号, 红色代表Gck阳性信号, 蓝色代表细胞核
Figure 8. Subcellular localization of G6pc and Gck in the liver of largemouth bass
Green indicates G6pc positive staining, red indicates Gck positive staining, blue indicates nuclear staining
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图 9 饲料淀粉水平对大口黑鲈糖代谢的影响
A. 血糖水平; B. 肝糖原含量(n=3); C. 基因表达(n=3); D. 蛋白质免疫印迹(n=3); E. 蛋白质免疫印迹量化分析; 数据以平均值±SD 表示(n=3); 不同字母代表不同处理之间存在显著差异
Figure 9. Effects of dietary starch level on glycometabolism in LMB
A. Blood glucose; B. Hepatic glycogen content (n=3); C. Gene expression (n=3); D. Western blot (n=3); E. Quantitative analysis of Western blot (n=3); Data are presented as means±SD (n=3); Different letters indicate significant differences among treatments
表 1 实验饲料配方及营养组成(干物质)
Table 1 Formulation and nutritional composition of the experimental diets (dry matter basis)
原料Ingredient (%) S8 S14 S20 木薯淀粉Cassava starch 7.00 14.00 19.00 微晶纤维素Microcrystalline cellulose 6.85 6.00 1.69 沸石粉Zeolite powder 6.84 1.69 0.00 其他Others1 79.3 79.3 79.3 营养组成Proximate composition (Analyzed, % dry matter) 水分Moisture 7.93 9.85 9.64 粗蛋白Crude protein 49.1 49.3 49.8 粗脂肪Crude lipid 11.4 11.6 11.6 淀粉Starch 8.13 14.1 20.1 灰分Ash 18.3 13.6 12.0 注: 1鱼粉. 34.0; 豆粕. 7.50; 谷朊粉. 15.0; 鸡肉粉. 13.0; 鱼油. 2.40; 豆油. 2.40; 磷酸二氢钙. 2.00; 多维多矿. 2.00; 维生素C磷酸酯. 0.20; 蛋氨酸. 0.25; 氯化胆碱. 0.50; 防腐剂. 0.01; 抗氧化剂. 0.05Note: 1 Fish meal. 34.0; soybean meal. 7.50; wheat gluten. 15.0; chicken meal. 13.0; fish oil. 2.40; soybean oil. 2.40; monocalcium phosphate. 2.00; vitamin and mineral premix. 2.00; VC phosphate. 0.20; methionine. 0.25; choline chloride. 0.50; antiseptic. 0.01; antioxidant. 0.05 
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表 2 引物序列
Table 2 Primer sequences
名称Name 引物序列Primer sequences (5′—3′) 退火TM (℃) 产物Product (bp) 截短表达载体构建Truncated expression vector construction pGEX-G6pc-F CGGGATCCATGAACGCTATAATGGACACCATGC 60.0 380 pGEX-G6pc-R CCGCTCGAGTTACATAGCATGTCCAGAGGGGCTG pGEX-Gck-F CGGGATCCATGCCGTGTGTCTCCTCTCAT 60.0 1394 pGEX-Gck-R CCGCTCGAGTTACTCCGACTGGATGAAGGTG 实时荧光定量PCR Quantitative Real-time PCR g6pca.1-F ACTTCCTTCGCTGTTGGCTT 60.5 150 g6pca.1-R ACCCATGTTACGCAGTAGGC g6pca.2-F TTCCCGCTTTGGTTTCACCT 61.0 195 g6pca.2-R AGTCTCACAGGTCATGGGGT gck-F CTCGCTCTGCTCGTATGT 55.0 207 gck-R CTCCCTTCCTCCGACTG pepck-F ACGTCAACTGGTTCCGCAAGA 65.0 142 pepck-R TGAGGCAGGTAGCCCACAGC pfkla-F TGCGTGGAGGGAGTTTTG 62.0 176 pfkla-R CTTGTGTCCGTGAGCGAG pk-F CGCTCTGCCCAGGATGTCAAAG 65.0 142 pk-R CAACCATCACGCCGTCACTCTC gys-F TTATTCTCTCTCGCTCCCG 62.0 150 gys-R TCTGCCTATCACCTGCCTC pygl-F AACCAACGGCATCACTC 58.5 144 pygl-R AGGGCAGCATTATCAACA glut2-F CTGGAAGAGATGAGGAGAG 57.5 155 glut2-R GAGCGGATAAACAGAATG eef1a1-F GTTGCTGCTGGTGTTGGTGAG 60.0 156 eef1a1-R GAAACGCTTCTGGCTGTAAGG 注: g6pc. 葡萄糖-6-磷酸酶催化亚基; gck. 葡萄糖激酶; pepck. 磷酸烯醇丙酮酸羧激酶; pfk. 磷酸果糖激酶; pk. 丙酮酸激酶; gys. 糖原合酶; pygl. 糖原磷酸化酶; glut. 葡萄糖转运蛋白; eef. 真核翻译延伸因子Note: g6pc. glucose-6-phosphatase catalytic; gck. glucokinase; pepck. phosphoenolpyruvate carboxykinase; pfk. phosphofructokinase; pk. pyruvate kinase; gys. glycogen synthase; pygl. phosphorylase glycogen, liver; glut. glucose transporter; eef. eukaryotic translation elongation factor
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