2015, Volume 17, Issue 1
Strategic Study of CAE >> 2015, Volume 17, Issue 1
Prokaryotic expression and bioactivity analysis of growth hormone and insulin-like growth factor-I from Platichthys stellatus
1. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266071, China;
2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
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Abstract
The mature peptide domain of insulin-like growth factor-I from Platichthys stellatus were amplified with specific primers based on its cDNA sequence. Then the matured peptide fragment was subcloned into the prokaryotic expression vector pET-28a to successfully construct IGF-I/pET-28a recombinant plasmid which were highly expressed in E.coli BL21（DE3） after being induced by IPTG with special fusion polypeptides containing His6 at their N-terminus. The obtained IGF-I polypeptide expressed in form of inclusion bodies with molecular weight of 12.1 kD and maximally accounted for 39.8 % of the whole bacterial protein post 3 h induction with 0.5 mmol/L IPTG at 37 ℃. Western blotting analysis indicated fusion polypeptides had the antigenicity to His6 antibody. The inclusion bodies were denaturalized using 6 mol/L guanidine HCl， purified using Ni-NTA affinity chromatography and annealed by gradient dialysis in urea， then purified proteins with molecular weight of 12.1 kD which was obtained from IGF-I recombinant bacterium. The proliferation experiment showed recombinant IGF-I protein could significantly promote the proliferation of human embryonic kidney cells HEK293T at 0.6 μg/mL and inhibit the proliferation with 1.8 μg/mL which verified its biological activity. Therefore， the IGF-I prokaryotic expression system was successfully constructed in the present study and biologically active IGF-I fusion protein was obtained. The present results would be helpful for better understanding the roles of IGF-I in growth regulation and development of high effective additive for growth promotion of Platichthys stellatus.
Keywords
Platichthys stellatus ; insulin-like growth factor-I ; prokaryotic expression ; bioactivity
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References
[ 1 ] Ponce M，Infante C，Funes V，et al. Molecular characterization and gene expression analysis of insulin-like growth factors I and II in the redbanded seabream，Pagrus auriga：transcriptional regulation by growth hormone [J]. Comparative Biochemistry and Physiology Part B：Biochemistry and Molecular Biology，2008， 150（4）：418-426. link1
[ 2 ] Peterson B C，Waldbieser G C，Bilodeau L. Effects of recombinant bovine somatotropin on growth and abundance of mRNA for IGF-I and IGF-II in channel catfish（Ictalurus punctatus）[J]. Journal of Animal Science，2005，83（4）：816-824. link1
[ 3 ] Picha M E，Turano M J，Beckman B R，et al. Endocrine biomarkers of growth and applications to aquaculture：A minireview of growth hormone，insulin- like growth factor（IGF）- I， and IGF- binding proteins as potential growth indicators in fish [J]. North American Journal of Aquaculture，2008，70（2）：196- 211. link1
[ 4 ] Reinecke M. Insulin-like growth factors and fish reproduction [J]. Biology of Reproduction，2010（82）：656-661. link1
[ 5 ] Maures T，Chan S J，Xu B，et al. Structural，biochemical，and expression analysis of two distinct insulin-like growth factor I receptors and their ligands in zebrafish [J]. Endocrinology，2002， 143（5）：1858-1871. link1
[ 6 ] 张俊玲，施志仪，付元帅，等. 牙鲆变态中 IGF-I 基因表达及甲 状腺激素对其的调节作用[J]. 水生生物学报，2011，35（2）： 355-359. link1
[ 7 ] Sitjà-Bobadilla A，Calduch-Giner J，Saera-Vila A，et al. Chronic exposure to the parasite Enteromyxum leei（Myxozoa：Myxosporea）modulates the immune response and the expression of growth， redox and immune relevant genes in gilthead sea bream，Sparus aurata L [J]. Fish & Shellfish Immunology，2008， 24（5）：610-619. link1
[ 8 ] Mccormick S D. Endocrine control of osmoregulation in teleost fish [J]. American Zoologist，2001，41（4）：781-794. link1
[ 9 ] Degger B G，Richardson N，Collet C，et al. In vitro characterization and in vivo clearance of recombinant barramundi（Lates calcarifer）IGF-I [J]. Aquaculture，1999，177（1）：153-160. link1
[10] Chen J，Chen J，Chang C，et al. Expression of recombinant tilapia insulin-like growth factor- I and stimulation of juvenile tilapia growth by injection of recombinant IGFs polypeptides [J]. Aquaculture，2000，181（3）：347-360. link1
[11] 叶 星，白俊杰，简 清，等. 草鱼胰岛素样生长因子-I基因在 大肠杆菌中的表达[J]. 中国生物化学与分子生物学报，2001， 17（6）：725-728. link1
[12] 张为民，张利红. 虹鳟胰岛素样生长因子I和Ⅱ在大肠杆菌中 的融合表达及促有丝分裂活性[J]. 动物学报，2003，49（2）： 266-271. link1
[13] 赵晓杰，陈松林，王 娜，等. 大菱鲆胰岛素样生长因子-I 成熟 肽的克隆，重组表达及活性分析[J]. 水产学报，2010（1）：1-7. link1
[14] Li Y，Wu S，Ouyang J，et al. Expression of insulin-like growth factor- 1 of orange- spotted grouper（Epinephelus coioides）in yeast Pichia pastoris [J]. Protein Expression and Purification， 2012，84（1）：80-85. link1
[15] 马爱军，庄志猛，李 晨，等. 星突江鲽生物学特性及养殖前景 [J]. 海洋水产研究，2006，27（5）：91-95. link1
[16] 刘振华，王 波，姚振刚，等. 星斑川鲽仔，稚，幼鱼的形态发 育与生长[J]. 海洋科学进展，2008，26（1）：90-97. link1
[17] Tian X C，Chen M J，Pantschenko A G，et al. Recombinant Epeptides of pro- IGF- I have mitogenicactivity [J]. Endocrinology，1999，140：3387-3390. link1
[18] 杨 辉，张英起，颜 真，等. 人血管形成素在大肠杆菌中的融 合表达、纯化及活性测定[J]. 生物工程学报，2001，17（1）：55- 58. link1
[19] Amersham Pharmacia Biotech. GST Gene Fusion System [M]. Third Edition. Revision 2. Pharmacia Biotech，Inc，2001.
[20] 叶 星，白俊杰，劳海华，等. 草鱼胰岛素样生长因子-I的融合 表达、纯化和抗血清制备[J]. 水产学报，2002，26（2）：122- 126. link1
[21] 刘芝亮，徐永江，柳学周，等. 半滑舌鳎类胰岛素生长因子-I的 原核表达及活性分析[J]. 中国水产科学，2013，20（4）：1-7. link1
[22] 杜 敏，朱美君，张莹夫，等. 钙蛋白酶抑制蛋白功能结构域 IV在大肠杆菌中的表达、纯化及其抗血清的制备[J]. 中国生 物化学与分子生物学报，2000，16（1）：23-27. link1
[23] 刘 侃，汪 炬，谢秋玲，等. 重组类胰岛素样生长因子-I 的纯 化与复性[J]. 中国生物工程杂志，2006，26（2）：29-33. link1
[24] Van Reeth T，Dreze P L，Szpirer J，et al. Positive selection vectors to generate fused genes for the expression of His- tagged proteins [J]. Biotechniques，1998，25：898-904. link1
[25] Hu S，Wu J，Huang J. Production of tilapia insulin-like growth factor-2 in high cell density cultures of recombinant Escherichia coli [J]. Journal of Biotechnology，2004，107（2）：161-171. link1
[26] 曹诣斌，吴兰亲，邵邻相，等. 青海湖裸鲤类胰岛素生长因子 IGF-Ⅱ的原核表达[J]. 水生生物学报，2010（2）：459-462. link1
[27] Fu P，Thompson J A，Leeding K S，et al. Insulin like growth factors induce apoptosis as well as proliferation in LIM 1215 colon cancer cells [J]. Journal of Cellular Biochemistry，2007，100 （1）：58-68. link1
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