MSTN基因克隆、表达及其主动免疫研究
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摘要
肌肉生长抑制素(Myostatin,MSTN)是一种抑制骨骼肌生长和发育的负向调控因子,其在肌细胞的生成过程中有着重要的功能。MSTN基因的突变或者人为的添加MSTN的抗体都能够提高动物的体增重,促进肌肉的发育(Mcpherron et al. 1997);口服表达抗原的酵母可以刺激机体产生特异性的抗体(Stubbs A C 2001)。本研究通过在酵母中表达MSTN基因,口服免疫小鼠,促进了小鼠的生长;并构建了MSTN整合的酵母菌株。
A.应用外显子拼接技术从猪基因组DNA中扩增出全长猪MSTN基因并构建了原核表达载体pET32a-MSTN。在大肠杆菌BL(DE3)中优化MSTN诱导表达条件并诱导表达带His标签的MSTN融合蛋白,通过镍层析柱纯化MSTN融合蛋白。
B.应用外显子拼接技术从鸡基因组DNA中扩增出全长鸡卵清蛋白(ovalbumin, OVA)基因并构建包含多克隆位点的酵母表达载体JMB88-OVA。从载体pET32a-MSTN酶切出MSTN基因插入用相同的内切酶消化过的JMB88和JMB88-OVA,构建酵母表达载体JMB88-MSTN和JMB88-OVA-MSTN。将构建好的两个酵母表达载体导入酵母JMY1中,硫酸铜诱导表达MSTN和OVA-MSTN。应用原核表达的MSTN蛋白和表达MSTN的酵母作为抗原,通过注射原核表达的MSTN蛋白和表达MSTN的酵母以及口服表达MSTN的酵母免疫生长期的小鼠;免疫7周后检测各组小鼠血清有无针对MSTN的抗体并分析小鼠的体增重和各主要骨骼肌重量,验证了口服免疫表达MSTN的酵母可以诱导小鼠产生针对MSTN的特异性的抗体,并促进小鼠生长和骨骼肌的发育。
C.在验证了口服表达MSTN的酵母有促进小鼠生长的功能后,设计并构建了MSTN的酵母整合载体pBlue-kan-NTS5’3’-MSTN。将线性化的pBlue-kan-NTS5’3’-MSTN载体导入酵母JMY1,获得MSTN整合酵母菌株JMY11;利用Cre/Loxp系统将报告基因KanMX4删除,得到无抗性基因的MSTN基因整合的酵母菌株JMY12;通过western blot验证了JMY12菌株可以持续表达MSTN蛋白。
本研究证明了口服表达MSTN的酵母可以诱导机体产生抗MSTN的抗体,并能够促进动物的生长;同时构建了安全的可直接用作饲料添加剂的表达MSTN的酵母菌。
Myostatin (MSTN) is a negative regulatory factor of skeletal muscle growth and development, which plays an important role in the generation of muscle cells. In this study, we amplified full-length porcine MSTN gene through assembly-exons and porcine MSTN was expressed in E.coli and purified after construction the prokaryotic expression vector of MSTN; Besides, we constructed MSTN yeast expression vectors and expressed MSTN in yeast (S.cerevisiae); We vaccinated mice by oral feeding with yeast expression MSTN and subcutaneous injection with yeast expression MSTN and MSTN protein to verify that oral yeast expression MSTN can induce mice to produce antibodies against MSTN. The antibodies against MSTN blocked physiological function of MSTN so as to promote muscle growth in mice; A marker-free recombinant yeast strain with porcine MSTN gene was constructed, which lays the foundation of industrial production for the yeast expression MSTN.
A. The full-length porcine MSTN gene was amplified by the method of assembly-exons from the pig genome, which was inserted into prokaryotic expression vector pET32a. The MSTN prokaryotic expression vector pET32a-MSTN was transformated into E.coli strain BL(DE3) and we optimized the condition of MSTN expression, expressed recombinant MSTN with His tag and purified MSTN fusion protein with nickel-chromatography.
B.We amplified full-length chicken ovalbumin (OVA) gene by the method of assembly-exons from chicken genome and constructed yeast expression vector JMB88-OVA. MSTN gene digested from pET32a-MSTN was inserted with the same enzymes digested JMB88 and JMB88-OVA to construct the MSTN yeast expression vector JMB88-MSTN and JMB88-OVA-MSTN. The two MSTN yeast expression vector were transformated into yeast strain JMY1 and expression of MSTN and OVA-MSTN was induced by CuSO4 and confirmed by western blot. We immunized mice by oral feeding with yeast expression MSTN or OVA-MSTN and subcutaneous injection with yeast expression MSTN or OVA-MSTN and MSTN protein. Serum samples of immunized mice were analyzed with western blot to validate that oral yeast can cause to humoral immune to produce antibodies against MSTN. The antibodies against MSTN can bind MSTN protein in mice body and block the MSTN function. Body weight gain and weight of the major skeletal muscle increased significantly in experimental groups comparing to control group.
C.After we investigated that it could promote muscle growth in mice orally immunized yeast expression MSTN. We designed and constructed a marker-free industrial yeast strain JMY12 integrated with MSTN cassette. Firstly we constructed the integration vector pBlue-kan-NTS5'3'-MSTN. The linearized integration vector pBlue-kan-NTS5'3'-MSTN was transformated into yeast JMY1 to integrate MSTN into yeast genome in which there was a marker gene KanMX4; Cre recombinase was transformated into the yeast integrated with MSTN and marker gene to delete KanMX4 gene at the use of Cre/Loxp system, and then we obtained the marker-free yeast strain integrated with MSTN gene, named JMY12; Expression of MSTN in JMY12 was verified by western blot.
This study demonstrated that mice fed with yeast expression MSTN could produce anti-MSTN antibodies so that the functions of MSTN would be neutralized and it promote the muscle growth in mice; we constructed a marker-free yeast expression MSTN.
A.应用外显子拼接技术从猪基因组DNA中扩增出全长猪MSTN基因并构建了原核表达载体pET32a-MSTN。在大肠杆菌BL(DE3)中优化MSTN诱导表达条件并诱导表达带His标签的MSTN融合蛋白,通过镍层析柱纯化MSTN融合蛋白。
B.应用外显子拼接技术从鸡基因组DNA中扩增出全长鸡卵清蛋白(ovalbumin, OVA)基因并构建包含多克隆位点的酵母表达载体JMB88-OVA。从载体pET32a-MSTN酶切出MSTN基因插入用相同的内切酶消化过的JMB88和JMB88-OVA,构建酵母表达载体JMB88-MSTN和JMB88-OVA-MSTN。将构建好的两个酵母表达载体导入酵母JMY1中,硫酸铜诱导表达MSTN和OVA-MSTN。应用原核表达的MSTN蛋白和表达MSTN的酵母作为抗原,通过注射原核表达的MSTN蛋白和表达MSTN的酵母以及口服表达MSTN的酵母免疫生长期的小鼠;免疫7周后检测各组小鼠血清有无针对MSTN的抗体并分析小鼠的体增重和各主要骨骼肌重量,验证了口服免疫表达MSTN的酵母可以诱导小鼠产生针对MSTN的特异性的抗体,并促进小鼠生长和骨骼肌的发育。
C.在验证了口服表达MSTN的酵母有促进小鼠生长的功能后,设计并构建了MSTN的酵母整合载体pBlue-kan-NTS5’3’-MSTN。将线性化的pBlue-kan-NTS5’3’-MSTN载体导入酵母JMY1,获得MSTN整合酵母菌株JMY11;利用Cre/Loxp系统将报告基因KanMX4删除,得到无抗性基因的MSTN基因整合的酵母菌株JMY12;通过western blot验证了JMY12菌株可以持续表达MSTN蛋白。
本研究证明了口服表达MSTN的酵母可以诱导机体产生抗MSTN的抗体,并能够促进动物的生长;同时构建了安全的可直接用作饲料添加剂的表达MSTN的酵母菌。
Myostatin (MSTN) is a negative regulatory factor of skeletal muscle growth and development, which plays an important role in the generation of muscle cells. In this study, we amplified full-length porcine MSTN gene through assembly-exons and porcine MSTN was expressed in E.coli and purified after construction the prokaryotic expression vector of MSTN; Besides, we constructed MSTN yeast expression vectors and expressed MSTN in yeast (S.cerevisiae); We vaccinated mice by oral feeding with yeast expression MSTN and subcutaneous injection with yeast expression MSTN and MSTN protein to verify that oral yeast expression MSTN can induce mice to produce antibodies against MSTN. The antibodies against MSTN blocked physiological function of MSTN so as to promote muscle growth in mice; A marker-free recombinant yeast strain with porcine MSTN gene was constructed, which lays the foundation of industrial production for the yeast expression MSTN.
A. The full-length porcine MSTN gene was amplified by the method of assembly-exons from the pig genome, which was inserted into prokaryotic expression vector pET32a. The MSTN prokaryotic expression vector pET32a-MSTN was transformated into E.coli strain BL(DE3) and we optimized the condition of MSTN expression, expressed recombinant MSTN with His tag and purified MSTN fusion protein with nickel-chromatography.
B.We amplified full-length chicken ovalbumin (OVA) gene by the method of assembly-exons from chicken genome and constructed yeast expression vector JMB88-OVA. MSTN gene digested from pET32a-MSTN was inserted with the same enzymes digested JMB88 and JMB88-OVA to construct the MSTN yeast expression vector JMB88-MSTN and JMB88-OVA-MSTN. The two MSTN yeast expression vector were transformated into yeast strain JMY1 and expression of MSTN and OVA-MSTN was induced by CuSO4 and confirmed by western blot. We immunized mice by oral feeding with yeast expression MSTN or OVA-MSTN and subcutaneous injection with yeast expression MSTN or OVA-MSTN and MSTN protein. Serum samples of immunized mice were analyzed with western blot to validate that oral yeast can cause to humoral immune to produce antibodies against MSTN. The antibodies against MSTN can bind MSTN protein in mice body and block the MSTN function. Body weight gain and weight of the major skeletal muscle increased significantly in experimental groups comparing to control group.
C.After we investigated that it could promote muscle growth in mice orally immunized yeast expression MSTN. We designed and constructed a marker-free industrial yeast strain JMY12 integrated with MSTN cassette. Firstly we constructed the integration vector pBlue-kan-NTS5'3'-MSTN. The linearized integration vector pBlue-kan-NTS5'3'-MSTN was transformated into yeast JMY1 to integrate MSTN into yeast genome in which there was a marker gene KanMX4; Cre recombinase was transformated into the yeast integrated with MSTN and marker gene to delete KanMX4 gene at the use of Cre/Loxp system, and then we obtained the marker-free yeast strain integrated with MSTN gene, named JMY12; Expression of MSTN in JMY12 was verified by western blot.
This study demonstrated that mice fed with yeast expression MSTN could produce anti-MSTN antibodies so that the functions of MSTN would be neutralized and it promote the muscle growth in mice; we constructed a marker-free yeast expression MSTN.
引文
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郭钦. 2009.食品级酿酒酵母高效分泌/展示表达系统构建. [博士学位论文].杭州:浙江大学
姜运良,李宁,杜立新. 2002.猪肌生成抑制素基因5’调控区T-A突变与生长性状的关系分析.遗传学报,29(5): 413~416
李绍华,熊远著,郑嵘. 2002.猪MSTN基因多态性及其SNPs的研究.遗传学报,29(4): 326~331.
刘娣,蒋国红,杨秀琴. 2001.东北民猪抑肌素基因cDNA的克隆和序列分析.见:陈宏权(主编).中国动物遗传育种研究进展第11次全国动物遗传育种学术会议论文集.北京:中国农业科技出版社: 31~34
刘俊云,张少恩,王秀梅,吴园园,唐海兵,郑伯建,孙娟. 2007.重组酵母疫苗载体技术的应用.中国生物制品学杂, 20(4):302~304
马现永,曹永长,舒鼎铭. 2004.肌肉生成抑制因子基因的克隆表达及动物免疫试验.中国科学,C辑, 34(6):522~526
欧阳红生,孙燕,张永亮. 2001.猪肌生成抑制素基因的克隆和序列测定.中国兽医学报,21(5):479~481
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