水貂IGF-Ⅰ基因的克隆、表达、功能检验及多态分析
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摘要
研究背景与目的:水貂是一种小型珍贵的毛皮动物,其经济价值主要在于它的毛皮是高档裘皮原料,对于水貂养殖业来说,皮张质量是经济效益的关键,而皮张的大小是衡量皮张等级的重要指标。因此,长期以来,培育出毛绒品质好、体型大、繁殖力强、生命力强的优良水貂,是人们坚持不懈的目标。动物的生长受生长轴的调控,生长轴是下丘脑—垂体—靶器官的一系列激素及其受体所组成的神经内分泌系统。其中GH—IGF-Ⅰ是生长轴的中心环节,在动物的生长调控中起着重要作用。GH的生物活性是通过IGF-Ⅰ来实现的。IGF-Ⅰ是一种对动物生长、发育、繁殖、营养代谢及组织细胞的增殖、分化、凋亡有直接影响的多功能生物活性肽。本研究以水貂为研究对象,开展了水貂IGF-Ⅰ基因的克隆、序列分析和原核表达、纯化,并将其作用于水貂成骨细胞观察其功能作用,探讨了今后生物工程生产水貂IGF-Ⅰ,用于提高水貂的生长性能的可能性。分析了IGF-Ⅰ基因exon1在6个水貂群体种中的多态性。
材料与方法:本研究根据小熊猫、金丝猴、大熊猫、人、华南虎、野马等物种的IGF-Ⅰ基因mRNA序列做参照,设计了扩增水貂IGF-Ⅰ基因cDNA序列。分析其成熟肽序列,结合表达载体pGEX-6P-1的特点设计、合成一对特异性引物。采用PCR的方法扩增水貂成熟IGF-Ⅰ蛋白cDNA片段,然后将其定向克隆到载体pGEX-6P-1中,构建原核表达重组载体pGEX-6p-1-IGF-Ⅰ;重组载体转入大肠杆菌DH5α扩增、提取后,用PCR、限制性内切酶酶切以及DNA序列测定等方法进行鉴定。鉴定正确的阳性pGEX-6p-1-IGF-Ⅰ重组质粒转化大肠杆菌BL21(DE3),通过Ampr抗性筛选获得稳定表达的阳性工程菌;以IPTG诱导工程菌高效表达重组蛋白IGF-Ⅰ,利用GST亲合层析、对重组IGF-Ⅰ蛋白进行分离纯化,并进行Western blot检测;最后通过体外细胞培养实验对重组IGF-Ⅰ蛋白进行功能的研究。另外以6个水貂群体共计360个个体为研究材料,采用PCR-SSCP、基因序列测定等方法分析了水貂IGF-Ⅰ基因exon1的多态性分布,对其进行了群体遗传学和统计学分析。
结果与结论:
1.利用RT-PCR方法从水貂肝脏组织扩增得到水貂IGF-Ⅰ的编码区序列,这个序列编码153个氨基酸的前体蛋白,包括48个氨基酸的信号肽、70个氨基酸的成熟肽、35个氨基酸的延伸肽。与GenBank中金丝猴、小熊猫、大熊猫、马等哺乳动物的IGF-Ⅰ序列比较,其编码成熟肽序列的同源性从牛(90%)到金丝猴(97%),开放阅读框序列的同源性从牛(91%)到金丝猴(96%)、小熊猫(96%)。
2.成功地构建了IGF-ⅠcDNA原核表达载体pGEX-6p-1-IGF-Ⅰ;建立了稳定转化pGEX-6p-1-IGF-Ⅰ重组质粒的大肠杆菌细胞株BL21-pGEX-6p-1-IGF-Ⅰ,并使IGF-ⅠcDNA在转化细胞内获得高效稳定的表达。确立了以可溶性形式高效表达重组IGF-Ⅰ蛋白的培养条件,较好地纯化出了IGF-Ⅰ蛋白,为以后进行更加深入的研究准备了基本条件。
3.成功的采用酶消化法和组织块贴壁综合的方法原代培养了水貂成骨细胞,结果表明所培养的细胞具有典型的成骨细胞形态特征,碱性磷酸酶染色、骨钙素染色、钙结节染色均呈阳性。证明培养出的细胞是成骨细胞。
4.不同剂量重组水貂IGF-Ⅰ蛋白对成骨细胞增殖和分化功能的影响,结果显示在0.01～0.8mg/ml的范围内能显著促进成骨细胞的增殖。
5.采用PCR-SSCP、基因序列测定等方法分析了水貂IGF-Ⅰ基因exon1的多态性分布,对其进行了群体遗传学和统计学分析。结果发现IGF-Ⅰ基因exon1内存在一处位点突变,位于185位核苷酸由C→G。
Background and Objectives: The mink is a small precious fur-bearing animal, whose main economic value is that its fur is the expensive raw material of making leather. For mink farming, the fur quality is critical for the economic benefit of farmers, and the size of a fur is a important index in evaluation of a fur. Therefore, our unremitting target is to breed this mink that has good fur quality, big body type, strong fecundity and strong vitality. Animal growth axis, regulates animal growth, is a neuroendocrine system which is made up of a series of hormones and receptors of hypothalamus-pituitary-target organ. GH-IGFⅠis a key component of growth axis, plays an important part in growth regulation. Bioactivation of GH is implemented by IGF-1 that is a multi-function peptide has a direct impact for animal growth, development, reproduction, nutrient metabolism and histiocytic multiplication, differentiation and apoptosis .The main contents of this study are that: firstly, implement gene clone, sequence analysis, prokaryotic expression and purification of IGF-1 in mink and observe its function in mink osteoblast; secondly, investigate bioengineering methods to produce IGF-1 of mink and employ it to improve growth performance of mink; finally, analyse the gene polymorphism of IGF-1 exonls in six mink populations.
Materials and Methods: Based on the lesser panda,golden monkey,giant panda,human etc,primer pairs for amplifying complete sequence of mink IGF-ⅠcDNA .The mature peptide was analyzed.It was cloned into pGEX-6p-1 plasmid through BamHI and EcoRI restriction site which was designated pGEX-6p-1-IGF-Ⅰ.The recombinant prokaryotic expression plasmid was confirmed by PCR amplification, restrictive enzymatic digestion and sequence analysis. Then the recombinant prokaryotic expression plasmid was transformed into E.coli. BL21(E3) cell. The stable transfectant, designated BL21-pGEX-6p-1-IGF-Ⅰ, was selected by the addition of Ampilin to the growth medium, followed by a series of confirmation. Bl21-pGEX-6p-1- IGF-Ⅰcells were cultured in LB medium till to OD600 value 0.6 to 0.8, then induced with IPTG to express recombinant IGF-Ⅰ, expression was detected by SDS-PAGE.The recombinant IGF-Ⅰexisted in a resoluble GST fusion protein form, its purification was employed by affinity chromatography column. The biological activity of recombinant IGF-Ⅰwas examined by mink osteoblastic cells growing tests in vitro.Otherwise, PCR-SSCP、DNA sequencing technique were applied in this study to analyze the distribution of IGF-Ⅰgene in 6 different mink populations,it is analyzed by population genetics and statistic genetics methods.
Results and Conclusion:
1. This study is aimed at Cloning,sequence analysis and expression、purification of IGF-Ⅰwhich playsed vital roles in growth,reproduction and milk secreting regulation.The significance and possibility of a further study towards the production of their recombinant IGF-Ⅰby recombinant DNA technique,and application in assisted growth and development.The main results were as the following:(1)By RT-PCR,liver IGF-Ⅰfrom mink,endoding 153 amino acids were isolated.,it comprised 48amino acids signal peptide ,70 amino acids mature peptide and 35 extend residues.(2)The mink IGF-Ⅰgene has a high homology with other mammal IGF-Ⅰgenes,and the homology of the sequence coding mature peptide is from 90%(cow)to 97%(golden monkey),ORF from 91%(cow)to 96%(golden monkey and lesser panda.
2.The recombinant prokaryotic expressing plasmid pgex-6p-1-IGF-Ⅰwas constructed successfully.Stable transfectants BL21-pGEX-6p-1-IGF-Ⅰwere obtained and the IGF-Ⅰwas overexpressed successfully. Stable transfectants BL21-pGEX-6p-1-IGF-Ⅰwere obtained and the IGF-Ⅰwas overexpressed successfully.
3. Successly cultured the osteoblast cells with enzyme digestion method and tissue adherent method in vitro.The results showed the cells obtained from mink costal bone were identified to be osteoblasts by inert microscope,ALP stain, immunochemistry stain of osteoblasts and alizarin red stain of Mineralization nodus. It indicated the cells obtained from mink costal bone showing typical osteoblasts’characteristics.
4.The effect of recombinant mink IGF-Ⅰprotein on the osteoblastic cells about proliferation was studied.The results showed certain concentrations of IGF-Ⅰcould stimulate the proliferation of theosteoblasts,and the former acted in dose-dependent manner in field of 0.01～0.8mg/ml.
5. PCR-SSCP、DNA sequencing technique were applied in this study to analyze the distribution of IGF-Ⅰgene in 6 different mink populations,it is analyzed by population genetics and statistic genetics methods.The results showed that one mutation site in exon1 of the mink IGF-Ⅰgene was detected,it was C→G at 185bp of the exon1.
材料与方法:本研究根据小熊猫、金丝猴、大熊猫、人、华南虎、野马等物种的IGF-Ⅰ基因mRNA序列做参照,设计了扩增水貂IGF-Ⅰ基因cDNA序列。分析其成熟肽序列,结合表达载体pGEX-6P-1的特点设计、合成一对特异性引物。采用PCR的方法扩增水貂成熟IGF-Ⅰ蛋白cDNA片段,然后将其定向克隆到载体pGEX-6P-1中,构建原核表达重组载体pGEX-6p-1-IGF-Ⅰ;重组载体转入大肠杆菌DH5α扩增、提取后,用PCR、限制性内切酶酶切以及DNA序列测定等方法进行鉴定。鉴定正确的阳性pGEX-6p-1-IGF-Ⅰ重组质粒转化大肠杆菌BL21(DE3),通过Ampr抗性筛选获得稳定表达的阳性工程菌;以IPTG诱导工程菌高效表达重组蛋白IGF-Ⅰ,利用GST亲合层析、对重组IGF-Ⅰ蛋白进行分离纯化,并进行Western blot检测;最后通过体外细胞培养实验对重组IGF-Ⅰ蛋白进行功能的研究。另外以6个水貂群体共计360个个体为研究材料,采用PCR-SSCP、基因序列测定等方法分析了水貂IGF-Ⅰ基因exon1的多态性分布,对其进行了群体遗传学和统计学分析。
结果与结论:
1.利用RT-PCR方法从水貂肝脏组织扩增得到水貂IGF-Ⅰ的编码区序列,这个序列编码153个氨基酸的前体蛋白,包括48个氨基酸的信号肽、70个氨基酸的成熟肽、35个氨基酸的延伸肽。与GenBank中金丝猴、小熊猫、大熊猫、马等哺乳动物的IGF-Ⅰ序列比较,其编码成熟肽序列的同源性从牛(90%)到金丝猴(97%),开放阅读框序列的同源性从牛(91%)到金丝猴(96%)、小熊猫(96%)。
2.成功地构建了IGF-ⅠcDNA原核表达载体pGEX-6p-1-IGF-Ⅰ;建立了稳定转化pGEX-6p-1-IGF-Ⅰ重组质粒的大肠杆菌细胞株BL21-pGEX-6p-1-IGF-Ⅰ,并使IGF-ⅠcDNA在转化细胞内获得高效稳定的表达。确立了以可溶性形式高效表达重组IGF-Ⅰ蛋白的培养条件,较好地纯化出了IGF-Ⅰ蛋白,为以后进行更加深入的研究准备了基本条件。
3.成功的采用酶消化法和组织块贴壁综合的方法原代培养了水貂成骨细胞,结果表明所培养的细胞具有典型的成骨细胞形态特征,碱性磷酸酶染色、骨钙素染色、钙结节染色均呈阳性。证明培养出的细胞是成骨细胞。
4.不同剂量重组水貂IGF-Ⅰ蛋白对成骨细胞增殖和分化功能的影响,结果显示在0.01～0.8mg/ml的范围内能显著促进成骨细胞的增殖。
5.采用PCR-SSCP、基因序列测定等方法分析了水貂IGF-Ⅰ基因exon1的多态性分布,对其进行了群体遗传学和统计学分析。结果发现IGF-Ⅰ基因exon1内存在一处位点突变,位于185位核苷酸由C→G。
Background and Objectives: The mink is a small precious fur-bearing animal, whose main economic value is that its fur is the expensive raw material of making leather. For mink farming, the fur quality is critical for the economic benefit of farmers, and the size of a fur is a important index in evaluation of a fur. Therefore, our unremitting target is to breed this mink that has good fur quality, big body type, strong fecundity and strong vitality. Animal growth axis, regulates animal growth, is a neuroendocrine system which is made up of a series of hormones and receptors of hypothalamus-pituitary-target organ. GH-IGFⅠis a key component of growth axis, plays an important part in growth regulation. Bioactivation of GH is implemented by IGF-1 that is a multi-function peptide has a direct impact for animal growth, development, reproduction, nutrient metabolism and histiocytic multiplication, differentiation and apoptosis .The main contents of this study are that: firstly, implement gene clone, sequence analysis, prokaryotic expression and purification of IGF-1 in mink and observe its function in mink osteoblast; secondly, investigate bioengineering methods to produce IGF-1 of mink and employ it to improve growth performance of mink; finally, analyse the gene polymorphism of IGF-1 exonls in six mink populations.
Materials and Methods: Based on the lesser panda,golden monkey,giant panda,human etc,primer pairs for amplifying complete sequence of mink IGF-ⅠcDNA .The mature peptide was analyzed.It was cloned into pGEX-6p-1 plasmid through BamHI and EcoRI restriction site which was designated pGEX-6p-1-IGF-Ⅰ.The recombinant prokaryotic expression plasmid was confirmed by PCR amplification, restrictive enzymatic digestion and sequence analysis. Then the recombinant prokaryotic expression plasmid was transformed into E.coli. BL21(E3) cell. The stable transfectant, designated BL21-pGEX-6p-1-IGF-Ⅰ, was selected by the addition of Ampilin to the growth medium, followed by a series of confirmation. Bl21-pGEX-6p-1- IGF-Ⅰcells were cultured in LB medium till to OD600 value 0.6 to 0.8, then induced with IPTG to express recombinant IGF-Ⅰ, expression was detected by SDS-PAGE.The recombinant IGF-Ⅰexisted in a resoluble GST fusion protein form, its purification was employed by affinity chromatography column. The biological activity of recombinant IGF-Ⅰwas examined by mink osteoblastic cells growing tests in vitro.Otherwise, PCR-SSCP、DNA sequencing technique were applied in this study to analyze the distribution of IGF-Ⅰgene in 6 different mink populations,it is analyzed by population genetics and statistic genetics methods.
Results and Conclusion:
1. This study is aimed at Cloning,sequence analysis and expression、purification of IGF-Ⅰwhich playsed vital roles in growth,reproduction and milk secreting regulation.The significance and possibility of a further study towards the production of their recombinant IGF-Ⅰby recombinant DNA technique,and application in assisted growth and development.The main results were as the following:(1)By RT-PCR,liver IGF-Ⅰfrom mink,endoding 153 amino acids were isolated.,it comprised 48amino acids signal peptide ,70 amino acids mature peptide and 35 extend residues.(2)The mink IGF-Ⅰgene has a high homology with other mammal IGF-Ⅰgenes,and the homology of the sequence coding mature peptide is from 90%(cow)to 97%(golden monkey),ORF from 91%(cow)to 96%(golden monkey and lesser panda.
2.The recombinant prokaryotic expressing plasmid pgex-6p-1-IGF-Ⅰwas constructed successfully.Stable transfectants BL21-pGEX-6p-1-IGF-Ⅰwere obtained and the IGF-Ⅰwas overexpressed successfully. Stable transfectants BL21-pGEX-6p-1-IGF-Ⅰwere obtained and the IGF-Ⅰwas overexpressed successfully.
3. Successly cultured the osteoblast cells with enzyme digestion method and tissue adherent method in vitro.The results showed the cells obtained from mink costal bone were identified to be osteoblasts by inert microscope,ALP stain, immunochemistry stain of osteoblasts and alizarin red stain of Mineralization nodus. It indicated the cells obtained from mink costal bone showing typical osteoblasts’characteristics.
4.The effect of recombinant mink IGF-Ⅰprotein on the osteoblastic cells about proliferation was studied.The results showed certain concentrations of IGF-Ⅰcould stimulate the proliferation of theosteoblasts,and the former acted in dose-dependent manner in field of 0.01～0.8mg/ml.
5. PCR-SSCP、DNA sequencing technique were applied in this study to analyze the distribution of IGF-Ⅰgene in 6 different mink populations,it is analyzed by population genetics and statistic genetics methods.The results showed that one mutation site in exon1 of the mink IGF-Ⅰgene was detected,it was C→G at 185bp of the exon1.
引文
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