Microsoft Word gc 刘传志

Transcription

1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 刘传志等 / 小鼠 17β-hsd10 的克隆 表达及双抗夹心 ELISA 方法的建立 November 25, 2014, 30 (11) : Chin J Biotech, All rights reserved 生物技术与方法 1775 小鼠 17β-hsd10 的克隆 表达及双抗夹心 ELISA 方法的建立 刘传志, 牛莹莹, 陈元安, 武成, 于源华 刘传志, 牛莹莹, 陈元安, 等. 小鼠 17β-hsd10 的克隆 表达及双抗夹心 ELISA 方法的建立. 生物工程学报,2014, 30(11): Liu CZ, Niu YY, Chen YA, et al. Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse. Chin J Biotech, 2014, 30(11): 摘要 : 原核表达小鼠 17β- 羟基类固醇脱氢酶 (17β-hsd10), 制备抗 17β-hsd10 多克隆抗体, 建立 17β-hsd10 双抗夹心酶联免疫检测方法 (Enzyme linked immunosorbent assay,elisa) 采用 RT-PCR 技术克隆得到小鼠肝脏 17β-hsd 10 基因, 构建原核表达体系 pet15b-17β-hsd10/ Escherichia coli BL21(DE3),IPTG 诱导重组蛋白表达, 并优化表达条件 ; 目的蛋白用 His 融合蛋白纯化柱 (His-Binding-resin) 纯化并免疫 BALB/c 小鼠和新西兰大耳白兔, 免疫血清总 IgG 采用硫酸铵沉淀法纯化, 用获得的高效价鼠源和兔源两种多克隆抗体, 建立 17β-hsd10 双抗夹心酶联免疫检测方法 表达蛋白约为 29.5 kda, 纯化后浓度为 1.5 mg/ml, 鼠源和兔源多克隆抗体效价分别为 和 , 建立的检测方法对于多种羟基类固醇脱氢酶无交叉反应, 特异性良好, 可检出含量约为 µg/ml 的阳性血清 建立的 17β-hsd10 双抗夹心 ELISA 方法, 简便 快速 敏感, 适合实验室研究科研广泛应用 并可能为检测阿尔茨海默病提供新思路和方法 : 17β- 羟基类固醇脱氢酶 10, 双抗夹心酶联免疫方法, 多克隆抗体 Received: January 26, 2014; Accepted: July 25, 2014 Supported by: Key Project in the Science and Technology Development Program of Jilin (No ). Corresponding author: Yuanhua Yu. Tel/Fax: ; yuyuanhua8888@126.com No

2 1776 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11 Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse Chuanzhi Liu, Yingying Niu, Yuan an Chen, Cheng Wu, and Yuanhua Yu School of Life Science and Technology, Changchun University of Science & Technology, Changchun , Jilin, China Abstract: We expressed 17β-hydroxysteroid dehydrogenase10(17β-hsd10) recombinant protein, prepared anti-17βhsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pet15b-17β-hsd10/escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/ml of 17β-hsd10 protein with molecular weight of 29.5 kda, and polyclonal antibodies from mouse and rabbit with the tite and respectively. The concentration of 0.1 µg/ml of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study. Keywords: 17β-hydroxysteroid dehydrogenase10, double-antibody Sandwich ELISA, polyclonal antibodies 17β- (17β-hsd10) (SDR) [1-3] 17β-hsd10 β [4-5] 17β-hsd10 3- A -II(SCHAD, HADH2) Aβ (ERAB) [6-8] 17β-hsd10 β- (Amyloid-β Aβ ) [5] [9-10] 17β-hsd10 Aβ Aβ [11] He (AD) 17β-hsd10 [12] Yang 17β-hsd10 AD 17β-hsd10 [13] 17β-hsd10 (Alzheimer's disease AD) [14-17] AD ( ) AD (MRI) (PET)

3 刘传志等 / 小鼠 17β-hsd10 的克隆 表达及双抗夹心 ELISA 方法的建立 1777 [18-19] AD 17β-hsd10 17β-hsd10 17β-hsd10 17β-hsd10 (ELISA) [20-21] µg/ml AD 1 材料与方法 1.1 主要材料 IPTG (Merck ) (Sigma ) His-Binding-resin ( ) BALB/c 96 (Costar) HRP DNM α-hsd 11β-hsd 17β-hsd4 6 Sigma -γ (MGL) pet-15b E. coli BL21(DE3) 1.2 方法 GenBank 17β-hsd10 NdeⅠ BamHⅠ ( ) β-hsd10 RNA 表 1 小鼠 17β-hsd10 基因引物序列 Table 1 Primer sequence of 17β-hsd10 gene in mouse Primer name Primer sequence (5 3 ) Forward Reverse Forward CATATGA is Nde BamH Ⅰ site. ACATATGATGGCTGCGGCAGTTCG GAGGGATCCTTAAGGCTGCATTCG Ⅰ site, reverse GGATCC is RNA RT-PCR 17β-hsd10 cdna PCR 94 5 min (94 2 min s 72 1 min) min 1% pucm-t (pucm-t-m10) pucm-t-m10 pet-15b NdeⅠ BamHⅠ 22 E. coli BL21 (DE3) PCR pet-5b-m10 37 ph 8.0 IPTG 0.5 mmol/l 48 h 12% SDS-PAGE (Ni) 17β-hsd β-hsd10 17β-hsd10 3 BALB/c ELISA P/N> r/min 5 min ( 50% ) ph 8.0 cjb@im.ac.cn

4 1778 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11 表 2 17β-hsd10 多克隆抗体免疫程序 Table 2 Immune Procedures of anti-17β-hsd10 polyclonal antibody The first immunization The second immunization The third immunization Rabbit 600 µg / rabbit, emulsified in complete Freund's adjuvant, injected hypodermically 600 µg / rabbit, emulsified in incomplete Freund's adjuvant injected intraperitoneally As above BALB/c mouse 200 µg /mus, emulsified in complete Freund's adjuvant injected intraperitoneally 200 µg /mus, emulsified in incomplete Freund's adjuvant injected intraperitoneally As above 2 3 IgG 4 5 ml ELISA ph9.6 17β-hsd µl/ 4 ( 0.05%<V/V>Tween 20 ph 7.0 PBS PBST) µl ( 10 mg/mlbsa) 37 1 h µl/ 37 1 h 3 17β-hsd µl/ 37 1 h 3 IgG 50 µl/ 37 1 h 3 ( TMB) 50 µl/ 15 min 2 mol/l 100 µl/ OD 450 S/N S/N= OD 450 / OD ELISA 3α-hsd 11β-hsd 17β-hsd4 7 His- -γ (MGL) β-hsd10 ( ) 100 µl ELISA 2 结果 β-hsd10 基因的克隆 RT-PCR 17β-hsd10 cdna pucm-t-m10 NdeⅠ BamHⅠ 789 bp β-hsd10 表达质粒的构建 PCR 2 pet-15b-m10 PCR 1 图 1 重组质粒 pucm-t-m10 双酶切鉴定电泳图谱 Fig. 1 Electrophoretogram is double enzyme digestion of pucm-t-m10. 1: samples; 2: pucm-t; M: DL5 000 DNA marker.

5 刘传志等 / 小鼠 17β-hsd10 的克隆 表达及双抗夹心 ELISA 方法的建立 1779 图 2 重组质粒 pet-15b-m10 双酶切鉴定及 PCR 鉴定电泳图谱电泳图谱 Fig. 2 Electrophoretogram is double enzyme digestion and PCR of pet-15b-m10. 1: PCR of pet-15b-m10; 2: sample; 3: pet-15b; M: DL5 000 DNA marker β-hsd10 的表达及纯化 IPTG 17β-hsd10 SDS-PAGE kda 2.4 双抗夹心 ELISA 方法的建立 17β-hsd µg/ml 1.0 µg/ml S/N ( 3) 图 3 17β-hsd10 蛋白诱导表达及纯化 SDS-PAGE 电泳图谱 Fig. 3 SDS-PAGE analysis of recombinant 17β-hsd10 protein expression and purified protein. M: marker ; 1, 2: 17β-hsd10; 3: E. coli BL21 (pet-15b); 4: E. coli BL21 (pet-15b-m10). 2.5 特异性实验 3α-hsd 11β-hsd 17β-hsd4 7 -γ (MGL) M10(+) 17β-hsd10 M10( ) 17β-hsd10 ( 4) 2.6 敏感性实验 17β-hsd10 (M10+) OD 450 ( 4) μg/ml 表 3 鼠源抗 17β-hsd10 多抗及兔源抗 17β-hsd10 多抗的最佳工作浓度确定 Table 3 Determination of the optimal concentration of coating antibody and rabbit-anti-17β-hsd10-igg Concentration of rabbit-anti-17β-hsd10-igg/(µg/ml) Concentration of mouse-anti-17β-hsd10-igg/(µg/ml) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.30 Carry 3 groups, two parallel samples of each group; the above data are average for each group the SD , SV 5.82% 8.38%. cjb@im.ac.cn

6 1780 ISSN CN /Q Chin J Biotech November 25, 2014 Vol.30 No.11 表 4 交叉反应实验 Table 4 The ELISA specific test Sample 3α-hsd 11β-hsd 17β-hsd4 17β-hsd7 MGL M10(+) M10( ) OD ± ± ± ± ± ± ±0.04 Carry 3 groups, two parallel samples of each group the above data are average for each group the SD , SV 5.32% 9.27%. Sample M10( ) 1:800 1: : : : : : : : OD ± ± ± ± ± ± ± P/N 图 4 ELISA 检测 M10 阳性血清的最大稀释倍数和 P/N 值 Fig. 4 The dilution ratio of the positive serum sample by ELISA. Carry 3 groups, two parallel samples of each group, the above OD 450 data are average for each group. 3 讨论 AD 17β-hsd10 17β-hsd10 BLAST 17β-hsd10 99% SDS-PAGE 17β-hsd10 E. coli 3α-hsd 11β-hsd 17β-hsd 17β-hsd µg/ml 17β-hsd10 17β-hsd10 ELISA 黙 REFERENCES [1] Lukacik P, Keller B, Bunkoczi G, et al. Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity. Biochem J, 2007, 402(3): [2] Mindnich R, Möller G, Adamski J, et al. The role of 17 beta-hydroxysteroid dehydrogenases. Mol Cell Endocrinol, 2004, 218(1/2): [3] Marchais-Oberwinkler S, Henn C, Möller G, et al. 17β-Hydroxysteroid dehydrogenases (17β-hsds)as therapeutic targets: protein structures, functions, and recent progress in inhibitor development. J Steroid Biochem Mol Biol, 2011, 125(1/2): [4] Hansis C, Jahner D, Spiess AN, et al. The gene for the Alzheimer-associated beta-amyloid-binding protein (ERAB)is differentially expressed in the testicular Leydig cells of the azoospermic by w/w(v)mouse. Eur J Biochem, 1998, 258: [5] He XY, Schulz H, Yu YS, et al. A human brain

7 刘传志等 / 小鼠 17β-hsd10 的克隆 表达及双抗夹心 ELISA 方法的建立 1781 L-3-hydroxyacyl-coenzyme a dehydrogenase is identical to an amyloid β-peptide-binding protein involved in Alzheimer s disease. J Biol Chem, 1998, 273(17): [6] Shafqat N, Marschall HU, Filling C, et al. Expanded substrate screenings of human and Drosophila type 10 17beta-hydroxysteroid dehydrogenases (hsds) reveal multiple specificities in bile acid and steroid hormone metabolism: characterization of multifunctional 3alpha/7alpha/7beta/17beta/ 20beta/ 21-H. Biochem J, 2003, 376(Pt 1): [7] Liao YC, Lee WJ, Hwang JP, et al. ABCA7 gene and the risk of Alzheimer's disease in Han Chinese in Taiwan. Neurobiol Aging, 2014, 10(1) [8] Rosenthal SL, Kamboh MI, et al. Late-onset Alzheimer's disease genes and the potentially implicated pathways.curr Genet Med Rep. 2014, 3(2): [9] He XY, Merz G, Yang YZ, et al. Characterization and localization of human type10 17betahydroxysteroid dehydrog-enase. Eur J Biochem, 2001, 268(18): [10] Yan SD, Fu J, Soto C, et al. An intracellular protein that binds amyloid-beta peptide and mediates neurotoxicity in Alzheimer s disease. Nature, 1997, 389(6652): [11] Hort J, O Brien JT, Gainotti G, et al. EFNS guidelines for the diagnosis and management of Alzheimer s disease. Eur J Neurol, 2010, 17(10): [12] He XY, Wen GY, Merz G, et al. Abundant type 10 17b-hydroxysteroid dehydrogenase in the hippocampus of mouse Alzheimer s disease model. Brain Res Mol Brain Res, 2002, 99(1): [13] Yang SY, He XY, Miller D, et al. Hydroxysteroid (17β) dehydrogenase X in human health and disease. Mol Cell Endocrinol, 2011, 343(1/2): 1 6. [14] Wu F, Cai ZY, Yan Y, et al. Advances in the pathogenesis of Alzheimer's disease metabolize sugars derived. Chongqing Medical, 2008, 37(17): (in Chinese).,,,.., 2008, 37(17): [15] Niu FN, Sun ZZ, Xu Y, et al. Advances in molecular targets marked the early diagnosis of Alzheimer's disease. J Inter Neurol Neurosurg, 2009, 1: (in Chinese).,,,.., 2009, 1: [16] Kong YY, Guan YH, Wu P, et al. Novel PET molecular probes for early diagnosis of Alzheimer s disease. Inter J Radiation Med Nuclear Medicine, 2012, 36(5): (in Chinese).,,,. PET., 2012, 36(5): [17] Rusinek H, Santi, Frid D, et al. Regional brain atrophy rate predicts future cognitive decline:6-year longitudinal MR imaging study of normal aging. Radiology, 2003, 229(3): [18] Dimou E, Booij J, Rodrigues M, et al. Amyloid PET and MRI in Alzheimer's disease and mild cognitive impairment. Curr Alzheimer Res, 2009, 6(3): [19] Valotassiou V, Archimandritis S, Sifakis N, et al. Alzheimer's disease: spect and pet tracers for beta-amyloid imaging. Curr Alzheimer Res, 2010, 7(6): [20] Clark CM, Schneider JA, Bedell BJ, et al. Use of florbetapir-pet for imaging beta-amyloid pathology. JAMA, 2011, 305(3): [21] Wong DF, Rosenberg PB, Zhou Y. In vivo imaging of amyloid deposition in Alzheimer Disease using the radioligand 18F-AV-45 (flobetapir [corrdcted] F 18). J Nucl Med, 2010, 51(6): ( ) cjb@im.ac.cn