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1 2015 9, 22(5): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J L 李树红, 陈志光, 李冉, 李新, 李松, 陈秀华, 蒋然然, 李美良, 马璐阳, 摘要 : L(Cathepsin L, CAT L) Ni-NTA, 300 mmol/l, SDS-PAGE TSK-GEL G2000SWxl CAT L, 28 kd, 95% Z-Phe-Arg-MCA CAT L, Cystatin 1.. 1, CAT L Balb/C, ELISA CAT L ; Western blotting, CAT L, CAT L CAT L : L; ; ; ; ; 中图分类号 : S965.1, TS254.1 文献标志码 : A 文章编号 : (2015) L(Cathepsin L CAT L), (C1 ), [1] CAT L, [2 3], [4] [5] [6] [7] [8],,, LPS, 鮰 (Ictalurus punctatus) [9] (Trachidermus fasciatus ) [10] (Oplegnathus fasciatus) (LPS ) [11] CAT L mrna, CAT L Ii MHCII, (Cyprinus carpio) L MHC Iclp-1 [12], 筴 (Decapterus maruadsi), CAT L, [13] CAT L mrna [14] [15], [15], CATs,, [16],, (Oncorhynchus keta), CAT L 3~7 [17],, [18] 收稿日期 : ; 修订日期 : 基金项目 : (2014NZ0003); (10ZA052). 作者简介 : (1975 ),,,,. lish@sicau.edu.cn

2 (Salmo salar) 4 d,, CAT L CAT B, [19], CAT L (Pseudosciaena crocea) CAT L mrna, TPA ph, [20], CAT L mrna,, CAT L, CAT L, CAT L (Cyprinus carpio var. jian),,, CAT L, CAT L,,,, CAT L mrna, CAT L [9 11, 21 22], CAT L,, CAT L,, CAT L (Cyprinus carpio var. jian) 500 g/,, Balb/C, (20±2) g, CAT L-pET-30a E.coli BL 21 (DE 3 ), Cystatin -β-d- (isopropyl-β-dthiogalactopyranoside, IPTG) ( ) ; Ni-NTA QIAGEN ; BCA ; Marker (14.4~94 kd) ; Z-Phe-Arg-MCA Sigma ; QuickAntibody Mouse 5W ; (GSH) (GSSG) IgG TMB OCT ; S-P DAB 1.2 Biologic Duo Flow Mini Protein3 PowerPac3000 Mini Trans-Blot Gel Doc 2000 ( Bio-Rad ); TSK G2000 SWXL ( TOSOH); LC-2010C HT ( SHIMADZU); Varioskan / ( Thermo Scientific ); ImageQuan ELC ( GE ); CX21 ( Olympus ); ( Leica ) 1.3 CAT L CAT L [23], CAT L-pET-30a E.coli BL 21 (DE 3 ),

3 5 : L 851, OD , 0.5 mmol/l IPTG, 37 2 h,, 1.4 CAT L CAT L [23], 0.5 mol/l 1 mol/l 8 mol/l, 8 mol/l ( 2 mmol/l GSH, 0.2 mmol/l GSSG, 0.5 mol/l NaCl, 1 mol/l 50 mmol/l Tris-HCl, ph 8.5) Ni-NTA, 20 mmol/l (phosphate buffer saline, PBS)(pH 7.4), CAT L CAT L, TSK-GEL G2000SWxl (7.80 mm 300 mm, 5 μm 125 Å), 10 μl, 0.1 mol/l Na 2 SO % NaN mol/l PBS, ph 6.7, 0.5 ml/min, 280 nm [25] 1.6 CAT L Cystatin Barrett [24] E-64 CAT L, 0.4 pmol Cystatin CAT L, CAT L Barrett [24], Z-Phe-Arg-MCA, ph 5.5, min, 380 nm, 460 nm 1.7 CAT L ELISA CAT L, [23] 1.8 CAT L CAT L, [23] 1.9 CAT L CAT L, [23] 1.10 SPSS 19.0, ( x SD ) CAT L CAT L( 1 2 ), ( 1 3), Ni 2+ -NTA, 300 mmol/l, ( 2), SDS- PAGE 28 kd ( 1 4) ( 3), 18.3 min, 96.3% V e V 0, 1 SDS-PAGE CAT L M: ; 1: pet-30a E.coli BL21 (DE 3 ) ; 2: CAT L-pET-30a E.coli BL21(DE 3 ) ; 3: ; 4: Ni 2+ -NTA. Fig.1 Expression and purification of recombinant CAT L detected by SDS-PAGE M: Standard protein marker; 1: The total protein of E.coli BL21 (DE 3 ) with transferred plasmid pet-30a; 2: The total protein of E.coli BL21 (DE 3 ) with transferred plasmid CAT L- pet-30a; 3: The sample washed with urea; 4: The sample purified with Ni 2+ - NTA affinity chromatography.

4 CAT L Ni 2+ -NTA Fig.2 Ni 2+ -NTA affinity chromatography of recombinant CAT L Cystatin 28.3 kd, SDS-PAGE 2.2 CAT L Cystatin 4, Cystatin, CAT L Cystatin 6 ng, CAT L 100% 36.30%,,, 0.4 pmol CAT L Cystatin 9.3 ng, Cystatin 21 kd [25], 0.44 pmol,, CAT L Cystatin 2.3 CAT L 1, ELISA, , P(+)/N( ) +, , P(+)/N( ), +, CAT L CAT L CAT L Western blotting CAT L 5, pet-30a, CAT L pet-30a-cat L, IPTG, CAT L,, CAT L,, CAT L, 2.5 CAT L CAT L, ( 6 ), CAT L CAT L, CAT L Fig. 3 item 3 TSK-GEL G2000SWxl CAT L Identification of recombinant CAT L protein by HPLC of TSK-GEL G2000SWxl Tab. 1 表 1 间接 ELISA 检测 CAT L 抗血清效价 Titer detection of anti-cat-l serum with indirect ELISA ( 10 3 ) dilution ratio of antiserum A 450 antiserum A A 450 serum A 450 before immune A 450 blank control A P(+)/N( )

5 5 : L 853 Fig. 4 4 CAT L Cystatin Titration curve of recombinant CAT L with Cystatin 5 Western blotting CAT L 1: pet-30a E.coli BL21(DE 3 ) ; 2: CAT L-pET-30a E.coli BL21(DE 3 ) ; 3: ; 4: Ni 2+ -NTA. Fig. 5 Prokaryotic expression of CAT L detected by Western blotting method 1: The total protein of E.coli BL21(DE 3 ) with transferred plasmid pet-30a; 2: The total protein of E.coli BL21(DE 3 ) with transferred plasmid CAT L-pET-30a; 3: The sample washed with urea; 4: The sample purified with Ni 2+ -NTA affinity chromatography. 3 CAT L, SDS-PAGE CAT L, CAT L Cystatin His, (Lampetra japonica) CAT L [21] (Cyclina sinensis) CAT L [26], (HUVEC) (ECV304) (MA), [21], [26] CAT L, CAT L, Western blotting CAT L,, Ahn [27] ELISA Western blotting, (Paralichthys olivaceus) CAT X CAT X CAT X,, CAT L,, CAT L, CAT L Aranishi [28] 25~29 kd CAT B, CAT B, (Gadus morhua) (Thunnus albacores) (Katsuwonus pelamis) (Pneumatophorus japonicus) CAT L, CAT L, CAT L,, CAT L, CAT L,,, CAT B CAT B,, CAT B ( ) [29] [30] (Fenneropenaeus chinensis) CAT L, CAT L

6 854 中国水产科学 图6 第 22 卷 免疫组化检测抗血清识别真核表达的 CAT L A. 小肠; B. 小肠绒毛; C. 骨骼肌细胞; D. 心肌; E. 脾; F. 肝胰脏; G. 骨骼肌. 箭头所示为阳性部位. Fig. 6 The detection of antiserum against CAT L expressed in eukaryotic using immunohistochemistry A. Small intestine; B. Intestinal villi; C. Skeletal muscle cell; D. Cardiac muscle; E. Spleen; F. Hepatopancreas; G. Skeletal muscle cell. The positive parts are indicated by arrows. 况; 对产卵洄游期大麻哈鱼白肌的免疫组化定位[31] 理功能和作用机制的研究均未见报道 本研究利 发现富含 CAT B 和 CAT L 活性的吞噬细胞可能 用原核 表达 制备了 具有 生物学 活性 的建鲤 重 组 [32] 参与了性成熟大麻哈鱼肌肉的深度软化 Zhou 等 CAT L 蛋白, 并制备了能够灵敏 特异地识别真 以刺参(Stichopus japonicus)类 CAT L 蛋白的多克隆 核 CAT L 的多克隆抗体, 为从蛋白水平监测建鲤 抗体, 利用免疫组化法发现 CAT L 在其体壁的表皮 生长发育过程中及在不同应激条件下 CAT L 的时 层分布比真皮层分布丰富得多, CAT L 分布密度与 空表达模式, 进而探索 CAT L 在鱼类生理病理中 海参体壁自溶速率呈现正相关性 的功能 作用机制, 奠定了良好的研究基础, 也为 目前, 关于基因和蛋白水平对建鲤 CAT L 生 基因水平上的深入研究提供了科学可靠的对比性

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9 5 : L 857 Prokaryotic expression, purification, characterization, and polyclonal antibody preparation of Jian carp (Cyprinus carpio var. jian) Cathepsin L LI Shuhong, CHEN Zhiguang, LI Ran, LI Xin, LI Song, CHEN Xiuhua, JIANG Ranran, LI Meiliang, MA Luyang College of Food Science, Sichuan Agricultural University, Ya an , China Abstract: The recombinant Cathepsin L (CAT L) protein of Jian carp (Cyprinus carpio var. jian) expressed in prokaryotic cells was washed by a gradient of urea concentrations and then purified by Ni 2+ -NTA agarose affinity chromatography. The target protein appeared as a single peak when eluted by 300 mmol/l imidazole in affinity chromatography. SDS-PAGE analysis and gel-filtration HPLC on a TSK-GEL G2000SWxl column revealed that recombinant CAT L was highly purified, and the molecular weight was about 28 kd with purity greater than 95%. The activity assay with Z-Phe-Arg-MCA as a substrate indicated that the recombinant CAT L could combine with its endogenesis inhibitor of Cystatin at a ratio, and thus took on the biological activity of cysteine protease. Balb/C mice were immunized by the purified protein to obtain antiserum, and ELISA showed that the titer of CAT L antiserum was higher than Western blotting showed that the CAT L polyclonal antibody was highly specific for recognizing recombinant CAT L protein expressed in prokaryotic cells. Immunohistochemistry analysis indicated that this antibody also recognized endogenous CAT L protein expressed in the hepatopancreas, muscle, small intestine, heart, and spleen of Jian carp. Based on these results, the polyclonal antibody obtained in this study could be used to detect CAT L expression and distribution in different tissues of fish based on protein level. Key words: Cathepsin L; prokaryotic expression; purification; characterization; polyclonal antibody; immunohistochemistry Corresponding author: LI Shuhong. lish@sicau.edu.cn