鲤胰岛素样生长因子的克隆与表达研究
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摘要
胰岛素样生长因子(Insulin-like growth factors，IGFs)，包括IGF-Ⅰ和IGF-Ⅱ，是一类具有胰岛素样代谢和促有丝分裂功能的单链多肽。可促进细胞的增殖、分化、迁移、生长和代谢。研究表明，IGFs在鱼类生长、繁殖、发育、渗透压调节及免疫等方面具有重要的作用。但由于血清中IGFs含量较低且与IGF结合蛋白结合存在，因此提取有活性的天然IGFs蛋白较困难。
为了获得足量的、具有生物学活性的重组鱼类IGFs蛋白，以利于鱼类IGF系统的基础和应用研究的不断深入，本研究在克隆鲤IGF-Ⅰ和IGF-Ⅱ的基础上，运用大肠杆菌原核表达系统和毕赤酵母真核表达系统对鲤IGF-Ⅰ和IGF-Ⅱ的基因工程表达进行了研究。
1．运用RT-PCR技术，从鲤肝脏总RNA中扩增出IGF-Ⅰ成熟肽cDNA序列，IGF-Ⅱ前肽cDNA序列，连接至pMD18-T载体，测序结果表明，与已报道的序列相比，克隆的鲤IGF-Ⅰ与其完全相同，IGF-Ⅱ基因有1个位点发生同义突变。
2．构建原核表达载体pGEX-KG-IGF-Ⅰm和pET-32a-IGF-Ⅱm，重组质粒转化大肠杆菌BL21并进行IPTG诱导表达，SDS-PAGE电泳分析，重组菌可表达分子量分别约为34kDa和28kDa的重组蛋白，且目的蛋白主要以包涵体的形式存在，经0.3 mmol／LIPTG诱导3h后，表达量均出现最高，占菌体总量的300％～35％。提取包涵体，并进行SKL变性及透析复性处理，由此初步纯化了原核重组蛋白IGFs。
3．以初步纯化的原核重组蛋白IGF-Ⅰ和IGF-Ⅱ为抗原，分别免疫新西兰大耳兔，制备了兔抗鲤IGF-Ⅰ／IGF-Ⅱ抗血清。抗血清经间接ELISA检测效价分别为3200和6400，Western-blot显示抗血清均能与重组蛋白发生特异性反应。
4．构建酵母表达载体pPIC9K-IGF-Ⅰm，LiCl法转化巴斯德毕赤酵母菌。经表型分析、G418筛选及PCR鉴定，得到His~+Mut~+型抗2.0 mg／mL G418的多拷贝转化子。以0.5％甲醇诱导培养，Dot-Blot及Tricine-SDS-PAGE电泳显示培养上清含大小约7.5kDa的重组IGF-Ⅰ蛋白，在诱导3d后，表达量最高。Western-blot分析表明，此真核表达蛋白能与兔抗IGF-Ⅰ多克隆抗体发生特异性免疫反应。
Insulin-like growth factors (IGFs), including IGF-Ⅰand IGF-Ⅱ, are single-chainpolypeptide hormones, which possess biological activity of growth stimulating activityand has similar metabolic functions of insulin. At the cellular level, IGFs could stimulatecell proliferation, differentiation, migration, metabolism, etc. Many studies show thatIGFs play very important regulated roles in the development, growth, reproduction,osmoregulation and immune of fishes. Because the concentration of IGFs in the extracellularenvironment is low and most of IGFs are bound to IGF-binding proteins, obtainingsufficient fish IGFs is very difficult.
In order to get sufficient quantity of biologically active piseine IGFs, This researchstudied the genetic engineering expression of IGFs in the E. coli and P. pastorisexpression systems at the base of cloning Common carp IGF-Ⅰand IGF-Ⅱ. What havebeen done in this research will be usefull for the continual progress of basic andapplication research of fish IGFs system.
1. Common carp IGFs gene was successfully amplified from liver total RNA withRT-PCR method, which was then cloned into vector pMD18-T and identified bysequencing. The sequencing result showed that: IGF-Ⅰwhich was cloned in this researchwas totally the same, compared with the sequences which had been reported in Genebank,and there was a synonymous mutation of 1 bp in IGF-Ⅱ.
2. Prokaryotic expression vectors pGEX-KG-IGF-Ⅰm and pET-32a-IGF-Ⅱm wereconstructed. After identified, the vectors were transformed into E. coli BL21 cell, andthen induced by IPTG. SDS-PAGE showed that each kinds of the recombinant stainscould express the fusion proteins (GST-IGF-Ⅰand Trx-IGF-Ⅱ) about 32 and 28 kDa, bothof the recombinant protein expressed in the form of inclusion body. After induced for 3hours by 0.3 mmol/ L IPTG, the yield was the highest, and the recombinant proteinamounted to 30%～35% of the whole protein in the E. coli cell. The method containingwith inclusion body collection-SKL denaturalization-dialysis renaturalization was used topurify these two prokaryotic expression proteins.
3. The purified product was used as the antigen to immunize New Zealand rabbit, thenthe rabbit anti-Common carp IGF-Ⅰ/IGF-Ⅱpolyclonal antibody was prepared. The titer ofthe antibody was 3200 and 6400 detected by ELISA. Western-blot suggested that both ofthe antiserums could react with corresponding recombinant protein specifically.
4. Yeast expression vector pPIC9K-IGF-Ⅰm was constructed. The vector waslinearizated, then transformed into the Pichia pastorist strain GS115 by LiCl. Theresearchers got recombinant strains with His~+Mut~+ phenotype which could resist2.0mg/mL G418, after phenotype analysis, G418 screening and PCR identification. The recombinant strains were induced by 0.5% methanol in shaking flasks. IGF-Ⅰwasexpressed and secreted into the supematant. When induced for 3 days, the yield was thehighest. Tricine-SDS-PAGE indicated that the recombinant protein was about 7.5 kDawhich was consistent with the expected result. Western-blot proved that the recombinantprotein could be specifically combined with rabbit antibodies to common carp IGF-Ⅰ.
为了获得足量的、具有生物学活性的重组鱼类IGFs蛋白，以利于鱼类IGF系统的基础和应用研究的不断深入，本研究在克隆鲤IGF-Ⅰ和IGF-Ⅱ的基础上，运用大肠杆菌原核表达系统和毕赤酵母真核表达系统对鲤IGF-Ⅰ和IGF-Ⅱ的基因工程表达进行了研究。
1．运用RT-PCR技术，从鲤肝脏总RNA中扩增出IGF-Ⅰ成熟肽cDNA序列，IGF-Ⅱ前肽cDNA序列，连接至pMD18-T载体，测序结果表明，与已报道的序列相比，克隆的鲤IGF-Ⅰ与其完全相同，IGF-Ⅱ基因有1个位点发生同义突变。
2．构建原核表达载体pGEX-KG-IGF-Ⅰm和pET-32a-IGF-Ⅱm，重组质粒转化大肠杆菌BL21并进行IPTG诱导表达，SDS-PAGE电泳分析，重组菌可表达分子量分别约为34kDa和28kDa的重组蛋白，且目的蛋白主要以包涵体的形式存在，经0.3 mmol／LIPTG诱导3h后，表达量均出现最高，占菌体总量的300％～35％。提取包涵体，并进行SKL变性及透析复性处理，由此初步纯化了原核重组蛋白IGFs。
3．以初步纯化的原核重组蛋白IGF-Ⅰ和IGF-Ⅱ为抗原，分别免疫新西兰大耳兔，制备了兔抗鲤IGF-Ⅰ／IGF-Ⅱ抗血清。抗血清经间接ELISA检测效价分别为3200和6400，Western-blot显示抗血清均能与重组蛋白发生特异性反应。
4．构建酵母表达载体pPIC9K-IGF-Ⅰm，LiCl法转化巴斯德毕赤酵母菌。经表型分析、G418筛选及PCR鉴定，得到His~+Mut~+型抗2.0 mg／mL G418的多拷贝转化子。以0.5％甲醇诱导培养，Dot-Blot及Tricine-SDS-PAGE电泳显示培养上清含大小约7.5kDa的重组IGF-Ⅰ蛋白，在诱导3d后，表达量最高。Western-blot分析表明，此真核表达蛋白能与兔抗IGF-Ⅰ多克隆抗体发生特异性免疫反应。
Insulin-like growth factors (IGFs), including IGF-Ⅰand IGF-Ⅱ, are single-chainpolypeptide hormones, which possess biological activity of growth stimulating activityand has similar metabolic functions of insulin. At the cellular level, IGFs could stimulatecell proliferation, differentiation, migration, metabolism, etc. Many studies show thatIGFs play very important regulated roles in the development, growth, reproduction,osmoregulation and immune of fishes. Because the concentration of IGFs in the extracellularenvironment is low and most of IGFs are bound to IGF-binding proteins, obtainingsufficient fish IGFs is very difficult.
In order to get sufficient quantity of biologically active piseine IGFs, This researchstudied the genetic engineering expression of IGFs in the E. coli and P. pastorisexpression systems at the base of cloning Common carp IGF-Ⅰand IGF-Ⅱ. What havebeen done in this research will be usefull for the continual progress of basic andapplication research of fish IGFs system.
1. Common carp IGFs gene was successfully amplified from liver total RNA withRT-PCR method, which was then cloned into vector pMD18-T and identified bysequencing. The sequencing result showed that: IGF-Ⅰwhich was cloned in this researchwas totally the same, compared with the sequences which had been reported in Genebank,and there was a synonymous mutation of 1 bp in IGF-Ⅱ.
2. Prokaryotic expression vectors pGEX-KG-IGF-Ⅰm and pET-32a-IGF-Ⅱm wereconstructed. After identified, the vectors were transformed into E. coli BL21 cell, andthen induced by IPTG. SDS-PAGE showed that each kinds of the recombinant stainscould express the fusion proteins (GST-IGF-Ⅰand Trx-IGF-Ⅱ) about 32 and 28 kDa, bothof the recombinant protein expressed in the form of inclusion body. After induced for 3hours by 0.3 mmol/ L IPTG, the yield was the highest, and the recombinant proteinamounted to 30%～35% of the whole protein in the E. coli cell. The method containingwith inclusion body collection-SKL denaturalization-dialysis renaturalization was used topurify these two prokaryotic expression proteins.
3. The purified product was used as the antigen to immunize New Zealand rabbit, thenthe rabbit anti-Common carp IGF-Ⅰ/IGF-Ⅱpolyclonal antibody was prepared. The titer ofthe antibody was 3200 and 6400 detected by ELISA. Western-blot suggested that both ofthe antiserums could react with corresponding recombinant protein specifically.
4. Yeast expression vector pPIC9K-IGF-Ⅰm was constructed. The vector waslinearizated, then transformed into the Pichia pastorist strain GS115 by LiCl. Theresearchers got recombinant strains with His~+Mut~+ phenotype which could resist2.0mg/mL G418, after phenotype analysis, G418 screening and PCR identification. The recombinant strains were induced by 0.5% methanol in shaking flasks. IGF-Ⅰwasexpressed and secreted into the supematant. When induced for 3 days, the yield was thehighest. Tricine-SDS-PAGE indicated that the recombinant protein was about 7.5 kDawhich was consistent with the expected result. Western-blot proved that the recombinantprotein could be specifically combined with rabbit antibodies to common carp IGF-Ⅰ.
引文
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87. Zinovieva N. Stable production of human insulin-like growth factor Ⅰ(hIGF-Ⅰ) in the milk of hemi and homozygous transgenic rabbits over sevsl generations. Transgenic Research, 1998, 7(6): 437-447
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74. Sakamato T, Hirano T. Expression of insulin- like growth factor Ⅰ gene in smoregulatory argans during sea water adaptation of the salmonid fish: possible mode of osmoregulatory action of growth hormone. Proc Natl Acad Sci USA, 1993, 90:1912-1916
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79. Shimizu M, Dickey J T, Dickhoff W W. Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-Ⅰ. JEndocrinol, 2005, 184:267-276
80. Skyrud T, Andersen O, Alestrom P, Gautvik K M. Effects of recombinant human growth hormone and insulin- like growth factor l on body growth and blood metabolites in brook trout (Salvelinus fontinalis). Gen Comp Endocrinol. 1989, 75(2): 247-255
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82. Upton Z, Yandeli C A, Degger B G, Chan S J, Moriyama S, Francis G L, Ballard F J. Evolution of insulin- like growth factor-i (1GF-I) action: in vitro characterization of vertebrate IGF-Ⅰ proteins. Comp Biochem Physiol B Biochem Mol Biol, 1998, 121: 35-41
83. Wallis A E, Devlin R H. Duplicated insulin-like growth factor Ⅰ genes in salflon display alternative splicing pathways. Mol Endocrinol, 1993, 7:409-422
84. Wilkinson R J, Elliott P, Carragher J F, Francis G. Expression, purification, and in vitro characterization of recombinant salmon insulin- like growth factor-Ⅱ. Protein Expr Purif, 2004, 35:334-343
85. Yada T, Nakanishi T. Interaction between endocrine and immune systems in fish. Int Rev Cytol, 2002, 220: 35-92
86. Zhang D C, Huang Y Q, Shao Y Q, Jiang S G. Molecular cloning, recombinant expression, and growth-promoting effect of mud carp (Cirrhinus molitorella) insulin-like growth factor-Ⅰ. Gen Comp Endocrinol, 2006, 148:203-212
87. Zinovieva N. Stable production of human insulin-like growth factor Ⅰ(hIGF-Ⅰ) in the milk of hemi and homozygous transgenic rabbits over sevsl generations. Transgenic Research, 1998, 7(6): 437-447