猪GDF11多克隆抗体的制备、前体脂肪细胞的分离培养及诱导分化研究
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摘要
本论文分为两个研究方向:猪GDF11蛋白表达产物的纯化及其多克隆抗体的制备与检测和猪前体脂肪细胞的分离培养及诱导分化。
第一部分:原核表达蛋白的纯化及多克隆抗体制备及检测
生长分化因子11(GDF11)是转化生长因子β(TGF-β)超家族、Myostatin/ GDF11亚家族的一种分泌蛋白。GDF11的表达水平可能与猪的胸腰椎数的多少有关。为了分析GDF11在不同胸腰椎数的猪品种中的表达差异,获得该蛋白的抗体十分必要。
将重组原核表达质粒pGEX-6P-1-GDF11经限制性内切酶BamHⅠ和EcoRⅠ双酶切,将回收的GDF11酶切片段插入原核表达载体pGEX-4T-2中,获得重组原核表达质粒pGEX-4T-2-GDF11。重组质粒经测序鉴定后转化大肠杆菌BL21(DE3),并经异丙基β-D-硫代半乳糖苷(IPTG)诱导产生了65KDa的GST-GDF11融合蛋白。融合蛋白以包涵体的形式存在,经稀释复性和凝血酶酶切,分离纯化出42 KDa左右的GDF11蛋白,SDS-PAGE分离纯化出GDF11蛋白。
纯化的GDF11蛋白注射小鼠,取其血清获得抗GDF11蛋白的多克隆抗体,经ELISA检测其滴度最高可达1∶2 5600。经Western印迹的结果表明,制备的多克隆抗体对LLC- PK1细胞内GDF11蛋白具有较高的特异性,是进一步研究GDF11表达调控机制和功能的基础。
第二部分:猪前体脂肪细胞的分离培养及诱导分化
脂肪沉积性状是猪的重要经济性状,与猪肉品质及其经济价值紧密联系。莱芜猪的肌间脂肪含量比其他品种都要高,研究其脂肪沉积有重要的意义。脂肪细胞在体外很难培养;而脂肪组织中的前脂肪细胞在体外可以培养,在一定条件下可诱导分化为脂肪细胞。
本研究从三日龄莱芜猪的背部皮下获得皮下脂肪,经胶原酶消化,分离培养了莱芜猪的前体脂肪细胞。前体脂肪细胞融合后的第二天,用胰岛素和地塞米松诱导分化,经诱导分化获得成熟脂肪细胞。在显微镜下观察细胞分化前后的形态,前体脂肪细胞呈纤维样;成熟的脂肪细胞变大、变圆,呈椭圆形或多边形。成熟的脂肪细胞经油红O染色,细胞内的脂滴被油红O着色。以成熟脂肪细胞的cDNA为模板,通过PCR的方法扩增LPL、PPARγ和C/EBPα,检测了这三个基因在成熟脂肪细胞中表达。本研究在细胞形态学和组织学上鉴定了分化的脂肪细胞,为分析脂肪分化的相关机制提供了重要的材料。
This thesis includes two parts: One is the expression and purification of growth /differentiation factor 11 (GDF11) and its polyclonal antibody preparation and detection. Another is the separation, culturing and induction to differentiation of porcine preadipocyte cells.
Growth/differentiation factor 11 is a secreted protein of myostatin/gdf11 subfamily of TGF-βsuperfamily. The expression level of GDF11 affects the number of thoracic and lumbar vertebra. Anti-GDF11 antibody is required for analyzing the relationship between the number of thoracic and lumbar vertebra and the expression level of GDF11.
After being digested with BamHⅠand EcoRⅠfor pGEX-6P-1-GDF11 expression plasmid, the GDF11 fragment was inserted into the prokaryotic expression vector pGEX-4T-2 and sequenced, and the recombinant plasmid pGEX-4T-2-GDF11 was obtained. The competent E.coli cell strain BL21(DE3) were transformed by the recombinant plasmid pGEX-4T-2-GDF11 and induced by IPTG to produce fusion protein 65KDa GST-GDF11. The expressed fusion protein GST-GDF11 was inclusion body. After diluting and renaturation, it was digested with thrombin to release 42KDa GDF11 protein. By SDS-PAGE, GDF11 protein is separated and purified.
The pure GDF11 proteins were injected into mouse to obtain anti-GDF11 polyclonal antibody. By ELISA detection, the antibody valency was 1:25600, and the antibody was shown to be specific by western blot analysis in porcine LLC- PK1 cell line, both of which meets the requirement for study on GDF11 function.
Fatty deposition is an important economical trait in pig breeding,and it has been shown to be closely related with pork quality and economic value. Comparing with other variations, intramuscular fat content in Laiwu pigs is very high, therefore, uncovering the genes for fat deposition in Laiwu pig is very important. Adipocytes is difficult to culture, but preadipocytes from adipose tissue can be isolated and cultured in vitro. Under special condition preadipocytes can be induced to lead differentiation and become mature adipocytes.
In this study, the subcutaneous fat from three 3-day-old Laiwu piglets was digested by Collagenase. Preadipocytes were isolated to culture from Laiwu pig adipose tissue. At the second day after preadipocytes cell fusion, we add trypsin and hexadecadrol in culture media to induce preadipocytes, then preadipocytes become to mature adipocytes. Preadipocytes show fibra shape under microscope, mature adipocytes become great and round , and adipocytes present ellipse and polygons. Mature adipocytes was stained by oil red O, and the lipid droplet present red. LPL, PPARγand C/EBPαwas amplified from mature adipocytes. In mature adipocytes cDNA, the genes were detected. We identify the mature adipocytes by morphology and histology. The mature adipocytes provide materials for analyzing the mechanism of preadipocyte differentiation.
第一部分:原核表达蛋白的纯化及多克隆抗体制备及检测
生长分化因子11(GDF11)是转化生长因子β(TGF-β)超家族、Myostatin/ GDF11亚家族的一种分泌蛋白。GDF11的表达水平可能与猪的胸腰椎数的多少有关。为了分析GDF11在不同胸腰椎数的猪品种中的表达差异,获得该蛋白的抗体十分必要。
将重组原核表达质粒pGEX-6P-1-GDF11经限制性内切酶BamHⅠ和EcoRⅠ双酶切,将回收的GDF11酶切片段插入原核表达载体pGEX-4T-2中,获得重组原核表达质粒pGEX-4T-2-GDF11。重组质粒经测序鉴定后转化大肠杆菌BL21(DE3),并经异丙基β-D-硫代半乳糖苷(IPTG)诱导产生了65KDa的GST-GDF11融合蛋白。融合蛋白以包涵体的形式存在,经稀释复性和凝血酶酶切,分离纯化出42 KDa左右的GDF11蛋白,SDS-PAGE分离纯化出GDF11蛋白。
纯化的GDF11蛋白注射小鼠,取其血清获得抗GDF11蛋白的多克隆抗体,经ELISA检测其滴度最高可达1∶2 5600。经Western印迹的结果表明,制备的多克隆抗体对LLC- PK1细胞内GDF11蛋白具有较高的特异性,是进一步研究GDF11表达调控机制和功能的基础。
第二部分:猪前体脂肪细胞的分离培养及诱导分化
脂肪沉积性状是猪的重要经济性状,与猪肉品质及其经济价值紧密联系。莱芜猪的肌间脂肪含量比其他品种都要高,研究其脂肪沉积有重要的意义。脂肪细胞在体外很难培养;而脂肪组织中的前脂肪细胞在体外可以培养,在一定条件下可诱导分化为脂肪细胞。
本研究从三日龄莱芜猪的背部皮下获得皮下脂肪,经胶原酶消化,分离培养了莱芜猪的前体脂肪细胞。前体脂肪细胞融合后的第二天,用胰岛素和地塞米松诱导分化,经诱导分化获得成熟脂肪细胞。在显微镜下观察细胞分化前后的形态,前体脂肪细胞呈纤维样;成熟的脂肪细胞变大、变圆,呈椭圆形或多边形。成熟的脂肪细胞经油红O染色,细胞内的脂滴被油红O着色。以成熟脂肪细胞的cDNA为模板,通过PCR的方法扩增LPL、PPARγ和C/EBPα,检测了这三个基因在成熟脂肪细胞中表达。本研究在细胞形态学和组织学上鉴定了分化的脂肪细胞,为分析脂肪分化的相关机制提供了重要的材料。
This thesis includes two parts: One is the expression and purification of growth /differentiation factor 11 (GDF11) and its polyclonal antibody preparation and detection. Another is the separation, culturing and induction to differentiation of porcine preadipocyte cells.
Growth/differentiation factor 11 is a secreted protein of myostatin/gdf11 subfamily of TGF-βsuperfamily. The expression level of GDF11 affects the number of thoracic and lumbar vertebra. Anti-GDF11 antibody is required for analyzing the relationship between the number of thoracic and lumbar vertebra and the expression level of GDF11.
After being digested with BamHⅠand EcoRⅠfor pGEX-6P-1-GDF11 expression plasmid, the GDF11 fragment was inserted into the prokaryotic expression vector pGEX-4T-2 and sequenced, and the recombinant plasmid pGEX-4T-2-GDF11 was obtained. The competent E.coli cell strain BL21(DE3) were transformed by the recombinant plasmid pGEX-4T-2-GDF11 and induced by IPTG to produce fusion protein 65KDa GST-GDF11. The expressed fusion protein GST-GDF11 was inclusion body. After diluting and renaturation, it was digested with thrombin to release 42KDa GDF11 protein. By SDS-PAGE, GDF11 protein is separated and purified.
The pure GDF11 proteins were injected into mouse to obtain anti-GDF11 polyclonal antibody. By ELISA detection, the antibody valency was 1:25600, and the antibody was shown to be specific by western blot analysis in porcine LLC- PK1 cell line, both of which meets the requirement for study on GDF11 function.
Fatty deposition is an important economical trait in pig breeding,and it has been shown to be closely related with pork quality and economic value. Comparing with other variations, intramuscular fat content in Laiwu pigs is very high, therefore, uncovering the genes for fat deposition in Laiwu pig is very important. Adipocytes is difficult to culture, but preadipocytes from adipose tissue can be isolated and cultured in vitro. Under special condition preadipocytes can be induced to lead differentiation and become mature adipocytes.
In this study, the subcutaneous fat from three 3-day-old Laiwu piglets was digested by Collagenase. Preadipocytes were isolated to culture from Laiwu pig adipose tissue. At the second day after preadipocytes cell fusion, we add trypsin and hexadecadrol in culture media to induce preadipocytes, then preadipocytes become to mature adipocytes. Preadipocytes show fibra shape under microscope, mature adipocytes become great and round , and adipocytes present ellipse and polygons. Mature adipocytes was stained by oil red O, and the lipid droplet present red. LPL, PPARγand C/EBPαwas amplified from mature adipocytes. In mature adipocytes cDNA, the genes were detected. We identify the mature adipocytes by morphology and histology. The mature adipocytes provide materials for analyzing the mechanism of preadipocyte differentiation.
引文
李明刚.高级分子遗传学.科学出版社,2004年.
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