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1 39 2 Vol. 39, No ACTA HYDROBIOLOGICA SINICA Mar., doi: 0.754/ 姜丽娟刘文生 2 林齐鹏饶飞翔杨惠暖王娟王涛 (., 50642; 2., 50642) 付晶晶 :, (Pelteobagrus fulvidraco), (PfGH) cdna, IPTG SDS-PAGE, Western IPTG.0 mmol/l 7 4h,,, 4, 35, (0.85±0.0) g, 3, 05 0 mg/l,, 0 4,, mg/l 29.67% (P<0.05).90% (P<0.05), 5 mg/l 2.32% (P<0.05) 8.83% (P<0.05), 4,, mg/l : ; ; ; ; 中图分类号 : S96.6 文献标识码 : A 文章编号 : (205) (Growth hormone, GH),,, [ 3] GH, [4] GH(rGH), GH, GH [5, 6] [7, 8] [9, 0] GH, GH [, 2], [3 6] 2, 6 65% 22% [7] ;, [0] ;, [8] ;, 4 [7],, GH, [3], [9] (Pelteobagrus fulvidraco) (Siluriformes), 鲿 (Bagridae), (Pelteobagrus), [20],,,,, [2], [22],,,, : 5.6 cm/5.7 g, 2 : 9.8 cm/20.6 g, 3 : 3.5 cm/36 g, 4 : 6 cm/58.2 g, 5 : 7.8 cm/8.3 g, 30 cm, ( ) g [23], 收稿日期 : ; 修订日期 : 基金项目 : (A200G02, A2030F03); (20) 作者简介 : (986 ), ; ; @qq.com 通信作者 : (965 ),, ; wsliu@scau.edu.cn

2 276 39,,,,,,. GH cdna ORF [24], 20, pet-28a(+), pet-pfgh GH cdna, PCR cdna, F: 5 -CTGGCTGCCAAGA TGATGGA-3, R: 5 -TCTCCACCTTGTGCATGTCC- 3, PCR,, pet-pfgh BL2(DE3),,,, 2% SDS-PAGE.2 GH 0.22 µm, 5 ml, Ni- His, Binding Buffer (20 mmol/l Tris-HCl, 0 mmol/l, 0.5 mol/l NaCl, ph 7.9), 0 /h, ; 5 Binding Buffer, ; mmol/l Elution Buffer (20 mmol/l Tris-HCl, 0.5 mol/l NaCl, ph 7.9),, SDS-PAGE 0 kd Millipore, r/min 0min,,,, Western, His, HRP IgG (H+L), BCA.3, (0.85±0.0078) g 80 cm 60 cm 50 cm, 35 4,, 3,, 3,,, ( /3),, 4% 6%, : 05 0 mg/l, 4, 45min ( ), 4, 4,, ( 24h), 5, 5, 5, 5,,, 80, ; (550 ), 05.5 (Survival rate, SR, %) =00 / (Weight gain rate, WGR, %)=00 (W t W 0 )/W 0 (Special growth rate, SGR, %/d) = 00 (Ln W t Ln W 0 )/t (Condition factor, CF, %)=00 / 3 (Viscerasomatic index, VSI, %) = 00 / (Hepaticsomatic index, HSI, %) = 00 /, W t W 0, t SPSS(V20) (One-way ANOVA), (P<0.05) Duncan s, ± (Mean± SE) 2 2. GH pet-pfgh BL2

3 2 : 277 (DE3), LB, PCR 424 bp, DNA,, GH cdna pet-28a, BL2, pet-pfgh-bl2 pet-pfgh-bl2 IPTG GH,, 2 kd 37 4h,, GH 7,, Ni- GH SDS-PAGE, 2 kd Western Blot,, SDS- PAGE, 7 GH,, 8.02 mg/ml GH 2.2 GH, 4, ( ), mg/l 5 mg/l (P<0.05), mg/l, 29.67%.90%, 4, ( 2) 2.3 GH GH 4,, ( 3) 3, ;,, 0 mg/l ; 4, ( 4) 2.4 GH GH ( 5) ( 6) Tab. catfish 表不同浓度 GH 浸泡对黄颡鱼幼鱼增重率特定生长率和存活率的影响 Effect of different concentrations of GH on the weight gain rate (WGR), specific growth rate (SGR) and survival of juvenile yellow GH WGR (%) SGR (%/d) SR (%) ±20.62 c 5.2±0.88 b 00± ±32.23 a 5.83±0. a 98.89± ±32.3 ab 5.67±0.2 a 00± ±39.5 bc 5.44±0.25 ab 99.29±.3 : 3 (P<0.05); Note: Data are the means of triplicate. Values with different superscripts have significant differences (P<0.05); the same applies bellow 表 2 生长激素停用后对黄颡鱼幼鱼增重率特定生长率和存活率的影响 Tab. 2 Effect of stopping the use of GH on the weight gain rate (WGR), specific growth rate (SGR) and survival of juvenile yellow catfish GH WGR (%) SGR (%/d) SR (%) ± ±0. 00± ± ± ± ± ±0.0 00± ± ±0.5 00±0.00 表 3 不同浓度 GH 浸泡对黄颡鱼幼鱼形态发育的影响 Tab. 3 Effect of different concentrations of GH on morphometric parameters of juvenile yellow catfish GH CF HSI VSI 0.75±0.04 ab.49±0.03 a 8.07±0.8.84±0.06 a.42±0.07 ab 7.97± ±0.07 ab.42±0.09 ab 7.35± ±0.09 b.23±0.02 b 7.20±0.6

4 Tab. 4 GH 表 4 生长激素停用后对黄颡鱼幼鱼形态发育的影响 Effect of stopping the use of GH on morphometric parameters of juvenile yellow catfish CF HSI VSI 0.75±0.02.5± ±0.3.78±0.07.6± ± ±0.05.6± ± ± ± ±0.09 表 5 不同 GH 浸泡浓度对黄颡鱼幼鱼体成分的影响 Tab. 5 Effect of different concentrations of GH on body compositions of juvenile yellow catfish (%) GH Moisture Crude protein Crude lipid ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.09 GH 表 6 不同 GH 浸泡浓度对黄颡鱼幼鱼体成分的影响 Tab. 6 Effect of stopping the use of GH on body compositions of juvenile yellow catfish (%) Moisture Crude protein Crude lipid 0 7.9± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.4 Ash Ash 3,, [25] GH cdna 603 bp, 200, 20, 80 GH,,, GH pet-28a, 6 His, [26], 2.27 kd, SDS-PAGE GH,,,, 37, 7, Ni-, His, GH,, [27 29] mg/l 5 mg/l 0 mg/l, mg/l,,,,,,, Kayes [30], [8] [8] Agellon [9], mg/l,,,,,,,,

5 2 : 279,, Wilson [3],,, [] Mclean E, Donaldson E M. Absorption of bioactive proteins by the gastrointestinal tract of fish: A review [J]. Journal of Aquatic Animal Health, 990, 2(): [2] Lebail P Y, Sire M F, Vernier J M. Intestinal transfer of growth hormone into the circulatory system of the rainbow trout Salmo gairdneri: interference by granule cells [J]. Journal of Experimental Zoology, 989, 25(): 0 07 [3] Moriyama S, Yamamoto H, Sugimoto S, et al. Oral administration of recombinant salmon growth hormone to rainbow trout (Oncorhynchus mykiss) [J]. Aquaculture, 993, 2(): [4] Lin T. Study on cloning, prokaryotic expression and biological activity of growth hormone gene of Schizothorax prenanti [D]. Thesis for Master of Science. Sichuan Agricultural University, Ya an [...,. 2008] [5] Ma X L, Zhang Y, Zhou L B, et al. Effects of LHRH-A on the growth and expressions of the growth axis in Nile tilapia Oreochromis niloticus [J]. Acta Hydrobiologica Sinica, 203, 40(): [,,,. LHRH-A., 203, 40(): 42 47] [6] Jiang S G, Zhang D C, Su T F, et al. Molecular cloning of growth hormone (GH) cdna from mud carp and the growth-promoting effects of the recombinant mcgh [J]. High Technology Letters, 2004, 4(6): [,,,. cdna., 2004, 4(6): 23 27] [7] Xu B. The growth-promoting effects of the recombinant eel growth hormone, methyl testosterone and thyroxine to pagrosomus major [C]. Shanghai: Tongji University Press. 995, [.,. :. 995, ] [8] Shi Z H, Zhou Y H. The effect of fish growth hormone on growth of black porgy, Acanthopagrus schlegeli (Bleeker) larva [J]. Modern Fisheries Information, 996, (): 3 6 [,.., 996, (): 3 6] [9] Chen R Z, Shuang B, Zhang R, et al. Recombinant fish growth hormone of Pichia pastoris as a feedstuff supplement [J]. Journal of Oceanography in Taiwan Strait, 2003, 22(2): [,,,.., 2003, 22(2): 80 86] [0] Jian Q, Bai J J, Ma J, et al. Research of enhancement of Tilapia growth by adding recombinant fgh in forage [J]. Freshwater Fisheries, 999, 29(3): 3 5 [,,,.., 999, 29(3): 3 5] [] Donaldson E M, Fagerlund U H M, Higgs D A, et al. Hormonal Enhancement of Growth [M]. Fish Physiology. New York: Academic Press. 979, [2] Mclean E, Donaldson E M. The endocrinology of growth, development and metabolism in vertebrates [C]. SanDiego: Academic Press. 993, 43 7 [3] Kawauchi H, Moriyama S, Yasuda A, et al. Isolation and characterization of chum salmon growth hormone [J]. Archives of Biochemistry and Biophysics, 986, 244(2): [4] Acosta J, Morales R, Morales A, et al. Pichia pastoris expressing recombinant tilapia growth hormone accelerates the growth of tilapia [J]. Biotechnology Letters, 2007, 29(): [5] Li Y H, Bai J J, Jian Q, et al. Expression of common carp growth hormone in the yeast Pichia pastoris and growth stimulation of juvenile tilapia (Oreochromis niloticus) [J]. Aquaculture, 2003, 26( 4): [6] Jeh H S, Kim C H, Lee H K, et al. Recombinant flounder growth hormone from Escherichia coli: overexpression, efficient recovery, and growth-promoting effect on juvenile flounder by oral administration [J]. Journal of Biotechnology, 998, 60(3): [7] Tsai H J, Lin K L, Kuo J C, et al. Highly efficient expression of fish growth hormone by Escherichia coli cells [J]. Applied and Environmental Microbiology, 995, 6(): [8] Zhang Q. Effects of recombinant-tuna growth hormone (R-TGH) on growth of Grass carp fingerling [J]. Journal of Fisheries of China, 993, 7(3): [.., 993, 7(3): ] [9] Agellon L B, Emery C J, Jones J M, et al. Promotion of rapid growth of rainbow trout (Salmo gairdneri) by a recombinant fish growth hormone [J]. Canadian Journal of Fisheries and Aquatic Sciences, 988, 45: 46 5l [20] Bai Y Q, Wang X, Liu D F, et al. The preferable light intensity and color for darkbarbel catfish and silver carp [J]. Acta Hydrobiologica Sinica, 204, 38(2): [,,,.., 204, 38(2): 26 22] [2] Zhen H H, Zhu X M, Han D, et al. Effects of dietary lipid level on growth and lipoprotein lipase gene expression in Pelteobagrus vachelli [J]. Acta Hydrobiologica Sinica, 200, 34(4): [,,,..

6 280 39, 200, 34(4): 85 82] [22] Dan C, Wang D, Gui J F. Chromosomal localization of sex-linked marker pf62-y in yellow catfish [J]. Acta Hydrobiologica Sinica, 204, 38(): [,,. Pf62-Y., 204, 38(): 84 86] [23] Zhang Z H. The biological characteristics of Pelteobagrus fulvidraco and its breeding and breeding technology [J]. Fisheries Economy Research, 2000, (4): 2 23 [.., 2000, (4): 2 23] [24] Sun Y H, Xie C X. Pelteobagrus fulvidraco growth hormone cdna sequence cloning and sequence analysis [J]. Reservoir Fisheries, 2006, 26(3): 0 3 [,. cdna., 2006, 26(3): 0 3] [25] Chen L. Analysis of the gene of Pseudosciaena crocea growth hormone and its expression in the Escherichia coli [D]. Thesis for Master of Science. Fuzhou University, Fuzhou [...,. 2004] [26] Ribas A V, Ho P L, Tanizaki M M, et al. High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli [J]. Biotechnology and Applied Biochemistry, 2000, 3(2): 9 94 [27] Collie N L, Stevens J J. Hormonal effects on L-proline transport in Coho salmo [J]. General and Comparative Endocrinology, 985, 59(3): [28] Sun L Z, Farmanfarmamian A. Biphasic action of growth hormone on intestinal amino acid absorption in striped bass bybrids [J]. Comparative Biochemistry and Physiology, 992, 03(2): [29] Fine M, Sakal E, Vashdi D, et al. Recombinant carp (Cyprinus carpio) growth hormone: expression, purification, and determination of biological activity in vitro and in vivo [J]. General and Comparative Endocrinology, 993, 89(): 5 6 [30] Kayes T. Effects of hypophysectomy, beef growth hormone replacement therapy, pituitary autotransplantation, and environmental salinity on growth in the black bullhead (Lctalurus melas) [J]. General and Comparative Endocrinology, 977, (33): [3] Wilson R P. Effect of recombinant bovine growth hormone administration on growth and body composition of channel catfish [J]. Aquaculture, 988, 73( 4): THE PREPARATION OF RECOMBINANT GROWTH HORMONE AND ITS EFFECTS ON THE GROWTH OF JUVENILE IN PELTEOBAGRUS FULVIDRACO JIANG Li-Juan, LIU Wen-Sheng, LIN Qi-Peng 2, RAO Fei-Xiang, YANG Hui-Nuan, WANG Juan, WANG Tao and FU Jing-Jing (. College of Animal Science, South China Agricultural University, Guangzhou 50642, China; 2. Public Basic Course and Experimental Teaching Center, South China Agricultural University, Guangzhou 50642, China) Abstract: In this study, the mature peptide-encoding cdna sequence of the growth hormone of Pelteobagrus fulvidraco (PfGH) was inserted into the prokaryote expression vector pet-28a (+). The recombinant plasmid of pet-pfgh was then transfected into Escherichia coli BL2 (DE3) and was induced by IPTG. The SDS-PAGE results showed that E. coli BL2 (DE3) with pet-pfgh expressed a specific fusion protein with a molecular weight of 2 KD. The western blot test revealed that the protein contained a 6-His tag. PfGH was abundantly expressed in E. coli cultured at 7 for 4 hours with the final IPTG concentration of.0 mmol/l. After ultrasonic disruption the soluble recombinant protein was recovered and purified, and was then applied to juvenile Pelteobagrus fulvidraco. The subjects with the average weight of (0.85±0.0) g were randomly divided into four groups and were raised in an indoor aquarium. Every group had three repeats with 35 fish in each. The experiments were conducted in the interior circulation water purification system. During the 4-week feeding trial, four groups were soaked in liquid containing the recombinant GH at different concentrations of 0 (the control), (group ), 5 (group 2) and 0 mg/l (group 3) respectively, and the soaking frequency was once a week. At the end of the trial, our results showed that compared to the control group, the weight gain rate (WG) and the specific growth rate (SGR) of group increased by 29.67% (P<0.05) and.90% (P<0.05) respectively; the WG and SGR of group 2 increased by 2.32% (P<0.05) and 8.83% (P<0.05) respectively. However, the body composition was not significantly different between the four groups. Furthermore, no significant difference was detected in the growth performance and the body composition between the four groups in 4 weeks after the cease of growth hormone treatment. Key words: Pelteobagrus fulvidraco; Growth hormone; Prokaryotic expression; Immersion; Weight gain rate

32期

32期中国农学通报 2014,30(32):38-42 Chinese Agricultural Science Bulletin 中草药饲料添加剂对黄鳝性激素含量的影响 1,2 1,2 1,2 2 赵庆凯, 丁炜东, 邴旭文, 王建新 ( 1 南京农业大学无锡渔业学院, 江苏无锡 214081; 2 中国水产