【作者】 王世凯；

【导师】 兰干球；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 肌肉生长抑制素（Myostatin）属于转化生长因子-β（TGF-β）超家族成员之一,又名为生长/分化因子-8（GDF-8）。Myostatin是在骨骼肌中广泛表达的一类糖蛋白,其作用特征是对肌肉生长有负调控作用,myostatin基因自然突变导致其生物活性的改变可以引起肌肉重量的显著增加。瘦素蛋白（Leptin）是由脂肪组织合成的一种重要肽类激素。通过与细胞膜上的长型受体结合,leptin启动了胞内一系列的信号转导系统,将身体能量贮存水平信号传送至大脑,从而对能量摄入与消耗进行调节。参照GenBank发表的猪myostatin和leptin基因序列,分别设计两对特异性引物,以广西巴马小型猪肌肉组织和脂肪组织的总RNA为模板,通过反向聚合酶链式反应（RT-PCR）扩增出相应的cDNA片段。扩增片段纯化后,连接到pMD18-T载体,转入DH5α细菌扩增并测序。结果表明:扩增的myostatin基因序列为1128bp,leptin成熟肽基因序列为441bp,包含了各自成熟肽序列。同源性比较显示,广西巴马小型猪myostatin和leptin基因与GenBank报道的猪的相应基因序列的同源性分别达到99.7%和99.1%。将克隆得到的myostatin和leptin基因序列,分别加入BamHI和EcoRI、BamHI和HindⅢ酶切位点,定向连接到原核表达载体pRSETA质粒上,构建pRSETA-MSNT₇₅和pRSETA-leptin融合表达载体,转化大肠杆菌BL₂₁（DE3）LysS。经SDS-PAGE和western Blotting分析,本研究成功地表达了myostatin和leptin的融合蛋白。表达蛋白经1 mmol/L IPTG诱导7h（myostatin）或4h（leptin）表达量达到最高,并以包涵体的形式存在。将pRSETA-MSNT₇₅和pRSETA-leptin重组菌进行大量表达后,包涵体用6mol/L的盐酸胍变性,可溶部分在50%Ni-NTA树脂上过柱纯化,通过透析使蛋白质复性,得到较纯的MSNT₇₅和leptin融合蛋白。利用纯化得到的融合蛋白制备了MSNT₇₅和leptin融合蛋白油乳剂疫苗。本研究应用广西巴马小型猪myostatin成熟肽重组融合蛋白疫苗主动免疫小鼠、广西巴马小型猪和鸡后,实验组和对照组相比,其平均体重和平均增重都有下降的趋势,但差异不显著（P＞0.05）,体内并未检测到相应的抗体。用广西巴马小型猪leptin成熟肽重组融合蛋白疫苗主动免疫小鼠后,小鼠的体重没有显著变化（P＞0.05）,但实验组采食量显著低于对照组（P＜0.05）。综上所述,本研究克隆了myostatin和leptin基因,成功表达了其成熟肽融合蛋白,制作了融合蛋白油乳剂疫苗,进行初步的动物免疫实验,所获得的结果为进一步研究myostatin和leptin基因的生物学功能及为myostatin和leptin基因在生产实践上的应用奠定了基础。更多还原

【Abstract】 Myostatin, also known as growth and differentiation factor 8, is a secreted glycoproteins and member of the transforming growth factor-βfamily that negatively modulates muscle satellite cell proliferation and inhibits muscle cell differentiation. Due to its properties, the naturally occurring mutations related to loss of function in the highly conserved myostatin gene lead to dramatic increase of muscle mass.Leptin is one of the most important adipose derived peptipe hormone which plays a key role in regulating energy intake and energy expenditure, including appetite and metabolism.Upon the binding to the long form leptin receptor to initiates multiple intracellular signal transduction pathways, leptin inform the brain about the status of the body’s energy store.In this study, primers were designed according the myostatin and leptin gene sequences released in GenBank. The total RNA purified from Guangxi Bama Mini-pig’s muscle and fat tissues, respevtively, was used to amplify myostatin and leptin cDNA by RT-PCR. The amplified segments were ligated into pMD18-T vector after purification and then the recombined vector was transformed into E.coli DH5αfor sequencing. The results indicated that the cloned myostatin and leptin cDNA contained 1128bp and 441bp, which included the coding sequence of entire length of myostatin and leptin mature peptides, respectively. Myostatin and leptin nucleodite sequence of Guangxi Bama Mini-pig shared 99.7% and 99.1% homologue to the previously released pig myostatin and leptin nucleodite sequence in Genbank, respectively.The cloned myostatin or leptin mature peptide cDNA was cloned into BamHI and EcoRI or BamHI and HindIII sites of expression vector pRSETA to construct the recombined plasmid pRSETA-MSNT₇₅ or pRSETA-Leptin. The recombined prokaryotic expression vectors then respectively transformed into BL₂₁（DE3）LysS to express myostatin or leptin fusion proteins after confirming the open reading frame was corrected. The proteins expressed were confirmed by SDS-PAGE and western blotting analysis. The expressing condition optimization indicated that the best cultivation temperature, IPTG concentration and inducing time for the fusion protein expression was 37℃, 1 mmol/L and 7h （myotatin） or 4h （leptin）, respectively.A sufficient amount of recombined myostatin or leptin fusion protein expressed in inclusion body form was denatured in guanidine hydrocloride （6mol/L） solution and purified by 50% Ni-NTA chromatography. The purified recombined fusion protein was refolded by dialysis and then homogenied with mineral oil to form immunogen used for animal immunization trail.Animals including mouse, Bama Mini-pigs and chickens were inoculated with the above prepared immunogens against myostatin. No antibody against recombined myostatin protein was detected in the immunized animal and no significant difference in body weight gain was observed between the immunized animals and the non-immunized animal （p>0.05）. Immunizing mouse with immunogen against leptin did not affected significantly on the average body weight gain（P>0.05）, but significantly drcrease animal’s feed intake （P<0.05）.It is concluded that immunizing animal with recombined myostatin protein did not yieded antibody and had not positive effect on the growth of animal, but immunizing animals with recombined leptin protein had positive effect on the performance of animals. The study lays the foundation for further study towards developing genetically-engineered myostatin and leptin vaccines.更多还原