水貂IGF-Ⅰ基因的克隆、序列分析及原核表达分析

中国生物工程杂志

2009, Vol. 29

Issue (08): 38-44

研究报告

水貂IGF-Ⅰ基因的克隆、序列分析及原核表达分析

荣敏¹,杨福合¹,许尚忠²,邢秀梅³,王桂武³,吴琼³

1. 中国农业科学院特产研究所吉林 1321092
2. 中国农业科学院北京畜牧兽医研究所北京 100094

Cloning,Sequencing Analysis and Prokaryotic Expression of the IGF-Ⅰ Gene from Mink

RONG Min¹, YANG Fu-He¹, HU Chang-Zhong², GENG Xiu-Mei¹, WANG Gui-Wu¹, WU Qiong¹

1. The Institute of Special Economic Animals and Plants, Chinese Academy of Agricultural Sciences,Jilin132109,China
2. Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing100094,China

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摘要：

获得水貂IGF-Ⅰ基因的cDNA序列，实现IGF-Ⅰ基因在大肠杆菌中的融合表达。利用RT-PCR技术从水貂肝脏组织扩增出水貂胰岛素样生长因子Ⅰ（IGF-Ⅰ）的编码区序列。此序列编码153个氨基酸的前体蛋白质。同源性分析表明IGF-Ⅰ的核苷酸和氨基酸序列与已报道的金丝猴、小熊猫、大熊猫、东北虎、马等哺乳动物的序列高度同源（＞90%）。对位比较发现水貂的信号肽序列具有氨基酸特异性，这些氨基酸是否影响水貂IGF-Ⅰ分子的构型、进而影响功能则需要进一步的研究。将构建的重组表达载体pGEX-6P-1-IGF-Ⅰ转入大肠杆菌BL21进行原核表达，得到高效融合表达，融合蛋白分子量约为34 KD。Western-blotting杂交证实了表达蛋白的抗原活性。本研究成功克隆、表达了水貂IGF-Ⅰ基因，分析和预测了其结构和功能，为进一步的生物活性研究打下基础。

关键词： 水貂胰岛素样生长因子-Ⅰ 克隆原核表达

Abstract:

The sequence of mink IGF-Ⅰgene was obtained and analyzed,and the fusion protein of the IGF-Ⅰ was produced in E.coli. By RTPCR,liver IGF-Ⅰfrom mink were isolated. The IGF-ⅠcDNAs encoding 153 amino acids,and showed high degree of homology(>90%)with other reported sequences(gold monkey,lesser panda,giant panda,A.tiger,horse,et al.) at nucleotide and amino acids levels.Two differences in mink amino acids sequences were found from sequence alignment.Whether these differences affect the molecular conformation or function for the growth factor's processing or these changes in the molecular structure may be responsible for the abnormal ability of growth and reproductive by influencing the function of IGF-Ⅰ have not yet been determined.The DNA fragment encoding the mature peptide of mink IGF-Ⅰwere subcloned to the pGEX-6P-1 expression vector and highly expressed in E.coli.BL21 with IPTG induction. The expression fusion proteins(pGEX-6P-1 IGF-Ⅰ)were mostly existed in soluble form and were about 34kDa.The induced cells culture were proved to be active IGF-Ⅰantigens. The mink IGF-Ⅰ gene was successfully cloned and expressed.Furthermore,the structure and function of this gene were analyzed and predicted. These result provided a good basis for further study of its biological activity.

Key words: Mink Insulin-like growth factor-Ⅰ Clone Prokaryotic expression

收稿日期: 2009-03-09 出版日期: 2009-07-28

通讯作者: 杨福合

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引用本文:

荣敏,杨福合,许尚忠,邢秀梅,王桂武,吴琼. 水貂IGF-Ⅰ基因的克隆、序列分析及原核表达分析[J]. 中国生物工程杂志, 2009, 29(08): 38-44.

RONG Min, YANG Fu-He, HU Chang-Zhong, GENG Xiu-Mei, WANG Gui-Wu, TUN Qiong. Cloning,Sequencing Analysis and Prokaryotic Expression of the IGF-Ⅰ Gene from Mink. China Biotechnology, 2009, 29(08): 38-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I08/38

［1］刘阳.蓝狐GDF8基因cDNA克隆及GDF8基因与IGFⅠ基因表达研究［学位论文］.东北林业大学.2005
Liu Y.A study on cDNA clone of GDF8 gene and the mRNA expression of GDF8 gene and IGF1 gene in blue fox.Harbin:Northeast Forestry University,2005
［2］ Daughaday W H，Hall K,Salmon W D,et al.J Clin Endocrinol Metab,1987,65:1075～1076
［3］ Froesch E R,Schmid C,Schwander J,et al.Am Rev Physiol,1985,47:443～467
［4］ Cohik W S, Clemmons D R. The insulin2like growth factors Ⅰ.Annu Rev Physiol, 1993, 55: 131
［5］ Jones J I,Clemmons D R.Insulinlike growth factors and their binding proteins:biological actions.Endocr Rev,1995,16:3～34
［6］ Upton Z,Yandell C A,Degger B G,et al.Evolution of insulinlike growth factorⅠ(IGFⅠ)action:in vitro characterization of vertebrate IGFⅠproteins.Comp Biochem Physiol B Biochem Mol Biol,1998,121:35～41
［7］ Lund P K,Moatsstaasts B M,Hynes M A,et al.Somatomedin C/insulinlike growth factor Ⅰand insulinlike growth factorⅡ mRNAs in rat fetal and adult tissues.J Biol Chem,1986,261:14539～14544
［8］ Von Heijne G. A new method for predicting signal sequence cleavage sites.Nucleic Acids Res,1986,14:4683～4690
［9］ Hodgson D R,May F E,Westley B R,et al.Involvement of phenylalanine 23 in the binding of IGFⅠ to the insulin and typeⅠIGF receptor.Regul Pept,1996,66(3):191～196
［10］ Cascieri M A,Chicchi G G,Applebaum J J,et al.Identification of the domain of IGFⅠresponsible for high affinity binding to the types 1 and 2 IGF receptors(IGFR1, IGFR2),insulin receptor(IR)and binding proteins(BP).FASEB J.1988,2:8577.
［11］ Nagagawa S H,Tger H S.Role of the COOHterminal Bchain domain in insulinreceptor interactions;identification of perturbations involving the insulin main chain.J Biol Chem,262(25):12054～12058
［12］ Bayne M L,Applebaum J,Chicchi G G,et al.The roles of tyrosines 24,31 and 60 in the high affinity binding of insulinlike growth factorⅠto the type 1 insulinlike growth factor receptor.J Biol Chem,1990,265:15648～15652
［13］胡细连.大熊猫等濒危动物IGFⅠ基因的克隆、表达与组织分布研究［学位论文］ .浙江大学，2005
Hu X L.Molecular Cloning,Expression and Tissue Distribution of Insulinlike Growth Factor Ⅰ in Endangered Animals.Hangzhou:Zhejiang University,2005
［14］ Stephen S,Ditchkoff Leon J Spicer,Ronald E,et al.Concentrations of insulinlike growth factorⅠin adult male whitetailed:associations with serum testosterone,morphometrics and age during and after the breeding season.Comparative Biothemistry and Physiology,2001， Part A 129:887～895
［15］ Moriyama S,Duguay S J,Conlon J M,et al. Recombinant coho salm on insulinlike growth factor Ⅰexpression in Escherichia coli,purification and characterization.Eur J Biochem,1993,218:205～211
［16］华益民，林浩然，等.草鱼IGFⅠcDNA的克隆和在原核生物中的表达.动物学报，2001，47（3）:274～279
Hua Y M,Lin H R.Cloning and expression in prokaryote of grass carp IGF Cdna.Acta Zoologica Sinica,2001,47(3):274~279
［17］张建峰.鸡IGFⅠ对成骨细胞功能的影响及其基因多态性与蛋鸡骨代谢相关性的研究［学位论文］.南京农业大学.2005
Zhang J F.Studies on effects of chicken IGFI on osteoblastic functions and its correlation of gene polymorphism to bone metabolism in cage layers.Nanjing:Nanjing Agricultural University,2005

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