【作者】 胡晓航；

【导师】 杨传平； 刘玉堂；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2005， 硕士

【摘要】 生长激素（Growth hormone,GH或somatotropin,ST）是动物垂体分泌的刺激动物生长的主要激素。随着脑垂体生长激素提纯技术的提高和基因重组技术的应用,成功的通过大肠杆菌高效地表达出牛、猪、鸡的生长激素,并已在奶牛、猪、鸡、羊等全面开展了注射生长激素效用的研究,表现出了明显的促生长作用。基因工程技术的诞生和发展,为大量生产生长激素提供了崭新的手段,利用基因工程手段,提高动物生长速度、饲料利用率及产品的回报率,同时也会在经济动物养殖及畜牧业等方面发挥重要作用。由此可见,GH在生产实践方面有广阔的应用前景。所以,不同种动物的GH纷纷在体外被克隆和表达出来,并试图进一步研究其生物学活性及其相关用途。 本研究的目的是通过克隆和表达银狐生长激素基因（fGH）,最终通过注射或投喂外源GH从而提高狐狸养殖的生产效率,促使银狐组织细胞分泌更多的生长激素,促进狐狸生长,生产出大幅张孤皮。我们采用传统的基因工程手段克隆到fGH基因并构建了其真核表达质粒pcDNA3.1-fGH和原核表达质粒pPROEX^TMHTa-fGH,并且都在宿主中得到了表达。这将促进对银狐生长激素结构与功能的基础研究,以及开发哺乳动物促生长剂有重要的意义,同时为外源生长激素直接注射狐狸骨骼肌后在体内的表达、持续时间及其对免疫功能的影响,进一步为研究GH在体内的作用奠定基础,同时也为GH基因治疗提供可能性。目前,在国内银狐的生长激素基因（fGH）的克隆和体外表达还未见研究报道,本研究即是从银狐脑垂体组织中分离生长激素基因,并进行克隆,然后再体外表达,为深入研究GH在生产实践中的应用提供材料和理论基础。 本实验的技术路线是分离并克隆银狐生长激素基因（fGH）,构建fGH基因的阳性重组质粒pMD18-T-fGH,然后再进行亚克隆,构建fGH成熟蛋白基因的原核表达质粒和真核表达质粒并分别转化大肠杆菌和转染雄性小鼠体内进行fGH成熟蛋白的表达。 为了获得具有生物活性的fGH蛋白,进一步探讨fGH更多的功能及对其应用前景的研究和开发,本实验构建了fGH基因的原核表达质粒pPROEX^TMHTa-fGH和真核表达质粒pcDNA3.1-fGH,并分别转化大肠杆菌和转染雄性小鼠体内,成功表达出fGH成熟蛋白。本实验方法是用Trizol的方法从银狐脑垂体组织中提取总RNA,以mRNA为模板,用RT-PCR方法扩增出生长激素成熟cDNA片段并克隆到载体质粒pMD18-T中,命名为pMD18-T-fGH;为了在真核细胞中表达fGH基因,我们设计上下游引物c₁、c₂,上游引物即c1含有fGH的5’端同源序列和HindⅢ酶切位点,在HindⅢ酶切位点后加入了起始密码子ATG,以启动蛋白质的翻译过程。下游引物即c₂含有3’端同源序列和EcoRI酶切位点。用c₁、c₂以pMD18-T-fGH为模板扩增fGH外源片段,用HindⅢ和EcoRI双酶切PCR扩增的fGH基因和真核表达载体pcDNA3.1（+）,并用DNA连接酶进行连接,转化大肠杆菌DH5α,提取质粒,并用酶切和PCR进行鉴定。将鉴定为阳性的真核表达载体质粒pcDNA3.1-fGH分别以10μg/100μl,100μg/100μl,250μg/100μl的不同浓度直接注射在小鼠的小腿肌肉中,与注射空载体pcDNA3.1（+）的对照组的小鼠作比较,每6天提取注射部位的肌肉组织,从中提取mRNA,然后进行反转录,得到cDNA,用Rt-PCR方法检测。发现质粒有表达。同时,通过含有BamHI酶切更多还原

【Abstract】 Growth hormone (GH or somatotropin, ST) is one of the most important hormones which stimulates animal to grow and excrete from pituitary. With the improvement of pituitary GH purification technology and the application of gene recombination, the bovine, pig and chicken GH which have been injected into milch cow, pig, chicken and sheep to develop their productive efficiency have been highly expressed through E . coil system and have informed obvious growth function. The development and born of gene project technology provide new method for mass production of GH. It has played a important role in improving animal growth rate, the using ratio of feed and production return, also in the aspect of economic animal breeding and stock raising. In a view, GH makes a promising application in the aspect of production and practice. According to this, various types of animal GH have been cloned and expressed out of body and researchers imagine farther investigating their biological functions and related use. At present, there is no report about sliver fox growth hormone (fGH) seen in our country. This experiment is to separate fGH gene from sliver fox pituitary issue and clone, express out of body. It will provide the bases on materials and theory for deeply invest on actual application of GH.Our objective is to gain fox growth hormone gene by cloning and expressing and finally to improve progenitive productivity, making more GH excrete from fox and increase its coat in size by injecting or feeding GH. The traditional gene project method was used and successfully constructed eukaryotic expression plasmid pcDNA3.1-fGH and prokaryotic expression plasmids pPROEXTMHTa-fGH. The result showed that both of them had expressed in the cell. It is significant in empoldering growth medicine and father study on possibility of fox GH gene therapy. At present, there is no report about sliver fox growth hormone (fGH) seen in our country. We separated fGH gene from sliver fox pituitary issue and clone, express out of body. It will provide the bases on materials and theory for deeply invest on actual application of GH.In order to gain biological functional fox growth hormone and deeply study its function and application, we constructed prokaryotic expressed plasmid of pPROEX ’ HTa-fGH transferred into E.coil and eukaryotic expressed plasmid of pcDNA3.1-fGH transfected into male rat. Then the mature protein was got successfully expressed. The method is that total RNA from fox pituitary issue is picked up with Trizol Kit. The specific primers of fGH gene which based on the sequence of the other canine strains was designed and synthesized. Specific fGH cDNA. 700bp, was amplified by RT-PCR with genomic RNA of fGH strain as the template for cDNA synthesis. PCR products were cloned into pMD18-T vector and named as pMD18-T-fGH. In order to express fGH gene in eukaryotic cell, according to the gene sequence of canine GH in Gene Bank, we designed a pair of primers c1, C2. The up primer c1 includes the 5’ region homologous sequence and the point of HindIII restriction enzyme; the down primer includes the 3’ region homologous sequence and the point of EcoRI restrictionenzyme. A 700bp-band was got by PCR with template of pMD18-T-fGH, after digested by restrict enzyme Hindlll and EcoRI. Then we ligated it into eukaryotic expressed vector pcDNA3.1 which digested by same enzyme. After identified by enzyme digestion and PCR, we transfected the positive recombinant eukaryotic expressed plasmid into calf muscles of male rats with following concentrations: 10u.g/100ul, lOOug/lOOul and 250ug/100ul Comparing with the rats which transfected pcDNA3.1 vector with nothing, we found the recombinant plasmid expressed through took the muscle issues of rats and picked up the total RNA then PCR every six days. Contemporary, we sub-cloned fGH gene cDNA segment with another special primer including the point of BamHI restriction enzyme by PCR and recombined it into prokaryotic vector pPROEX?HTa which was digested by restrict enzyme EcoRI and BamHI. After ligated, the prokaryotic expressed plasmid pPROEX?HTa-fGH was identified by enzymes digestion and PCR. Then the positive recombinant prokaryotic expressed plasmid was transferred into E.coil DH5 a . Under the inducement of final concentration lmmol/L IPTG, the high expressed amount of GH protein was detected by SDS-PAGE electrophoresis and it occupied total E.coil protein 35%.The recombinant protein in Ecoil DH5 a expressed in the form of excuse body. Cloning of fox GH gene and the expression whether in prokaryotic or eukaryotic system lay a foundation of farther study on the structure, biological function even operated machismo of fGH gene on the level of cell and molecule. In this way, we can make a deep research on the practice aspect of promoting growth in mammiferous GH gene.更多还原