广西巴马小型猪Myostatin和Leptin基因的克隆、表达及动物免疫实验
|
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-MSNT_(75)和pRSETA-leptin融合表达载体,转化大肠杆菌BL_(21)(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-MSNT_(75)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MSNT_(75)和leptin融合蛋白。利用纯化得到的融合蛋白制备了MSNT_(75)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。
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-MSNT_(75) or pRSETA-Leptin. The recombined prokaryotic expression vectors then respectively transformed into BL_(21)(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.
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-MSNT_(75)和pRSETA-leptin融合表达载体,转化大肠杆菌BL_(21)(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-MSNT_(75)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MSNT_(75)和leptin融合蛋白。利用纯化得到的融合蛋白制备了MSNT_(75)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。
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-MSNT_(75) or pRSETA-Leptin. The recombined prokaryotic expression vectors then respectively transformed into BL_(21)(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.
引文
[1]McPherron A C,Lawler A M,Lee S J,et al.Regulation of skeletal muscle mass in mice by a new TGF-βsuperfamily member.Nature,1997,387:83-90.
[2]Carlson C J,Booth F W,Gordon S E.Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading[J].American Journal of Physiology Regulatory Integrative and Comparative Physiology,1999,277:601-606
[3]Kocamis H,Killefer J.Myostatin expression and possible functions in animal muscle growth[J].Domestic Animal Endocrinology,2002,23:447-454.
[4]Roberts S B,Goetz F W.Differential skeletal muscle expression of myostatin across teleost species,and the isolation of multiple myostatin isoforms[J].FEBS Letters,2001,491(3):212-216.
[5]Ji S,Losinski R L,Cornelius S G.Myostatin expression in porcine tissues:tissue specificity and developmental and postnatal regulation.Am J Physiol,1998,275:R1265-R1273.
[6]Xu C,Wu G,Zohar Y,Du S J.Analysis of myostatin gene structure,expression and function in zebrafish[J].The Journal of Experimental Biology,2003,206:4067-4079.
[7]Radaelli G,Rowlerson A,Mascarello F,et al.Myostatin precursor is present in several tissues in teleost fish:a comparative immunolocalization study[J].Cell Tissue Res.,2003,311:239-250
[8]Salamonsen L A,Dimitriadis E,Robb L.Cytokines in implantation[J].Seminars in Reproductive Medicine,2000,18(3):299-310
[9]McPherron A C,Lee S J.Double muscling in cattle due to mutations in the Myostatin gene[J].Proc Natl Acad Sci USA,1997,94:12 457-12 461.
[10]Jeanplong F,Sharma M,Somers W G,etal.Genomic organization and neonatal expression of the bovine myostatin gene[J].Mol Cell Biochem,2001,220(1-2):31
[11]Stratil A,Kopecny M.Genomic organization,sequence and polymorphism of the porcine myostatin(GDF8,MSTN)gene[J].Anim Genet,1999,30(6):468-70.
[12]Sazanov A,Ewald D,Buitkamp J,et al.Amolecular marker for the chicken myostatin gene(GDF8)mapsto 7p11[J].Anim Genet,1999,30(5):388-9.
[13]Thomas M,Langley B,Berry C et al.Myostatin,a negative regulator of muscle growth,functions by inhibiting myoblast proliferation[J].Biol Chem,2000;275:40235-40243
[14]Grobet L.A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[J].Nat Genet,1997,17(1):71-74.
[15]Kambadur R,Sharma M Smith T P L,et al.Mutations in myostatin(GDF-8)in Dubled-muscled Belgian Blue and Piedmontese Cattle[J].PCR Methods Appl,1997,7(9):910-915.
[16]李绍华,熊远著,郑嵘,等.猪MSTN基因多态性及其SNPs的研究[J].遗传学报,2002,29(4):326-331.
[17]Lin J,Heather B.Myostatin knockout in mice increase myogenesis and decreses adipogensis[J].Bioch Biop Res Comm,2002,292(3):701-706.
[18]Jannel Acosta,Yamila Carpio,Ingrid Borroto,et al.Myostatin gene silenced by RNAi show a zebrafish gaint phenotype[J].Journal of Biotechnology,2005,119(4):324-331
[19]McPherron A C,Lee S J.Suppression of body fat accumulation in Myostatin deficient mice[J].The Journal of Clinical Investigation,2002,109(5):595-601.
[20]Kim H S,Liang L,Dean R G,Hausman D B,Hartzell D L,Baile C A.Inhibition of preadipocyte differentiation by myostatin treatment in 3T3-L1 cultures.Biochemical and Biophysical Research Communications,2001,281(4):902-906.
[21]Rebbapragada A,Benchabane H,Wrana J L,Celeste A J,Attisano L.Myostatin signals through a transforming growth factor-like signaling pathway to block adipogenesis.Molecular and Cellular Biology,2003,23(20):7230-7242.
[22]Reardon K A,Davis I.Myostatin,insulin-like growth factor-1,and leulemia inhibitoryfactor mRNAs are upregulated in chronic human disuse muscle atrophy[J].Muscle Nerve,2001,24(7):893-899.
[23]Nester F,Gonzalez-Cadevid,Wayne E,et al.Organization of the human myostatin gene and expression in healthy men of HIV-infected men with muscle wasting[J].Proc Natl Acad Sci USA,1998,95(25):14938-14943.
[24]Gilson H,Schakman O,Combaret L,et al.Myostatin gene deletion prevents glucocorticoid-induced muscle atrophy[J].Endocrinology,2007,148(1):452-460.
[25]Liu W,Thomas SG;Asa SL,Gonzalez-Cadavid N,Bhasin S,and Ezzat S.Myostatin is a skeletal muscle target of growth hormone anabolic action[J].J Clin Endocrinol Metab.2003,88(11):5490-5496.
[26]Mark Thomas,Brett Langley,CaroleBerry,et al.Myostatin,a negativeregulator of muscle growth,functions by inhibiting myoblast proliferation[J].J biol Chem,2000,275(51):40235-40243.
[27]Rios R,Carneiro I,Arce V M,et al.Myostatin is an inhibitor of myogenic differentiation[J].Am J Physiol Cell Physiol,2002,282(5):993-999
[28]Joulia D,Bernardi H,Garandel V,et al.Mechanisms involved in the inhibition of myoblast proliferation and differentiation by Myostatin[J].Exp Cell Res,2003,286(2):263-275
[29]Zhu X,Hadhazy M,Wehling M,et al.Dominant negative myostatin produces hypertrophy without hyperplasia in muscle[J].FEBS Lett,2000,474(1):71-75
[30]Rios R,Carneiro I,Arce VM,etal.Myostatin regulates cell survival during C2C12myogenesis[J].Biochem Biophys Res Commun,2001,280(2):561-566.
[31]Georges M,Grobet L,Pirottin D.Towards transgenic engineering of the myostatin gene in livestock.BiotechNet Proceedings,2004,1-4.
[32]陈晔华,张传茂,陈俭,等.分子细胞生物学[M].北京:清华大学出版社]2006:358-378
[33]Zhang Y,Proenca R,Maffei M,et al.Positional cloning of the mouse obese gene and its human homologue.Nature,1994,372:425-432.
[34]孙长颢,张宏伟,王舒然,等.不同膳食构成对大鼠肥胖基因表达的影响[J].中国公共卫生,1998,14(5):277-280
[35]Ingalls,A.M.,Dickie,M.M.& Snell,G.D.,OBese,a new mutation in the house mouse[J].Hered.1950,41,317-318.
[36]Hummel,K.P.,Dickie,M.M.,and L.,C.D..Diabetes,a new mutation in the mouse[J].Science,1966,153:1127-1128.
[37]Coleman D L.Effects of parabiosis of OBese with diabetes and normal mice.Diabetologia,1973,9:294-298.
[38]Tartaglia L,Dembski M,Wang X,et al.Identification and expression cloning of a leptin receptors,OB-R[J].Cell,1995,83:1263-1271.
[39]Friedman,J.M.,Halaas,J.L.,Leptin and the regulation of body weight in mammals,Nature,1998,395:763-770.
[40]R.S.Ahima,C.Prabakaran,C.Mantzoros,et al.Role of leptin in the neuroendocrine response to fasting[J].Nature.1996,382:250-252.
[41]屈会起,邱明才,林珊.瘦素与肾脏[J].国外医学内科学分册,2000,27(3):111-114
[42]Levin,et al.Decrease food intake does not completely account for adiposity reduction after ob protein infusion[J].Proc Natl Acad Sci USA,1996,93:1726-1730.
[43]Chen,et al.Disappearance of body fat in normal rats induced by adenovirus-mediated liptin gene therapy[J].Proc Natl Acad Sci USA,1996,93:14795-14799
[44]Pelleymounter MA,Cullen MH,Baker MB,et al.Effects of the obese gene product on body weight regulation in ob/ob mice[J].Science,1995,269:540-543.
[45]Vogel G.Leptin:a trigger for puberty.Science,1996,274(5292):1466-1467.
[46]JJ Hwa,AB Fawzi,MP Graziano,et al.Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice[J].Am J Physiol,1997,(8):1204-1209.
[47]Carro E,Pinila L,Seoalle LM,et al.Influence of endogenous leptin tone on the estrous cycle and luteinizing hormone pulsatility in female rats[J]Neuroendocrinology,1997,66:375-377
[48]Spicer L J,Francisco C C,The adipose obese gene product,lepting edidence of a direct inhibitory role in ovarian function[J].Endocrinology,1997,138:3374-3379.
[49]Brogan R S,Mitchel S E,Trayhum P,et al.Suppression of leptin during lacation:contribution of the suckling stimulus versus milk producfion [J].Endocrinology,1999,140(6):2621-2627.
[50]丛琳.肥胖蛋白受体.国外医学卫生学分册,2001,28(4):232-234
[51]吴坚,邹大进.瘦素受体的研究进展.生理科学进展.2000,31(2):143-146
[52]许梓荣,赵国芬,郑家茂.Leptin生物学功能的研究进展及应用前景[J].动物营养学报.2001,13(1):1-5
[53]郭亚宁,候水生,刘小林,等.Leptin的生物学功能研究进展[J].中国畜牧兽医,2002,29(5):26-29.
[54]Yu WH,Kimura M,Walczewska A,et al.Role of leptin in hypothalamic-pituitary function[J].Proc Natl Acad Sci USA,1997,94:1023-1028.
[55]Legradi G,Emerson CH,Ahima RS,et al.Leptin prevents fasting-induced suppression of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypothalamic paraventricular mucleus.Endocrinololgy,1997,138:2569-2576.
[56]兰干球,李芳芳,黄雄军,等.广西巴马小型猪肥胖基因(Leptin)成熟肽序列的cDNA的克隆、序列分析[J]。广西农业生物科学,2007,4
[57]Ma X Y,Cao Y C,Shu D M,et al.Cloning and expression of swine myostatin gene and its application in animal immunization trial.Science in China Ser.C Life Seciences,2005,48(4):368-374.
[58]姜运良,李宁,吴常信,等.不同品种猪肌肉生长抑制素基因单核苷酸多态性分析[J].遗传学报,2001,28(9):840-845
[59]RAMSAY T G,YAN X,MORRISON C,et al.The obesith gene in swine [J].Sci,1998,76(2):484-490.
[60]张冰,杨公社,孙超,等.Leptin基因对陆川猪和大白猪产仔数的影响[J].畜牧兽医学报.2007,38(6):608-613
[61]Lagonigro R,Wiener P,Pilla F,et al.A new mutation in the coding region of the bovine leptin gene associated with feed intake[J].Animal Genetics.2003,34:371-374.
[62]Kimchi-Sarfaty C,Oh J M,Kim I W,et al.A "Silent" polymorphism in the MDR1gene changes substrate specificity[J].Science,2007,315(5811:525-528
[63]Sharma M,Kambadur R,mattews K G,et al.Myostatin,a transforming growth factor-[beta]superfamily member,is expressed in heart muscle and is upregulated in cardiomyocytes after infarct[J].Cell Physiol,1999,180:1-9
[64]徐万祥,邱德义,顾少华,等.去N端信号肽和C端跨膜区猪卵透明带-3B 蛋白在原核系统表达的研究[J].生物工程学报,2001,7(1):16-19
[65]Karl R,Afzalpurkar A,Gupta S K,et al.Expression of bomet monky(Macaca radiata)Zona pellucida-3(zp3)in a prokatyotic system and its immunogenicity.Mol Reprod Dev[J].1997,47:140-147
[66]卢圣栋,《现代分子生物学实验技术》第2版[M],中国协和医科大学出版社,1999
[67]金元昌,刘利,苏晓艳,李景鹏,李会东,周建红IPTG诱导浓度、时间及温度对重组促性腺激素释放激素基因表达的影响[J]黑龙江畜牧兽医.2006,8:17-19
[68]何忠效,张树政.生物化学实验技术丛书—电泳 第2版[M],科学出版社,1999
[69]李春丽,陈其新,何国庆 植物甜蛋白des-pGlul-brazzein在大肠杆菌中表达[J].食品与发酵工业.2006,32(7):8-11
[70]卢圣栋,等.现代分子生物学实验技术第2版[M],中国协和医科大学出版社,1999
[71]蒲勤.人骨形成蛋白2成熟肽在大肠杆菌中的表达、纯化及其生物学活性的研究[M].第四军医大学博士学位论文,2000
[72]戴茹娟.猪肥胖基因(OB)和肥胖基因(OB-R)cDNA的克隆与分析[D].北京:中国农业大学,1998.
[73]J.萨姆布鲁,D.W.拉赛尔,等.《分子克隆实验指南》第三版[M].北京:科学出版社,2002:1682-1683
[74]于自然,黄熙泰,等.《现代生物化学》[M].北京:化学工业出版社,2001.
[75]黎敏义.猪抑制素α亚基重组融合蛋白的制备及应用研究[D].广州:华南农业大学,2006.
[76]许正平,段治军,陈长征,等.NMT在大肠杆菌中的His_6融合表达及其纯化研究.生物化学与生物物理学报.[J]1997,29(4):383-388
[77]陈长征,黄华,龚健,等.人肠杆菌His_6融合表达载体及其表达产物的一步纯化.[J]生物化学与生物物理学报.1996,28(5):523-529
[78]QIAGEN.A handbook for high-level expression and purification of 6×His-tagged proteins.[M]Fifth Edition,2003
[79]Maeda Y,Ueda T,Imoto T.Effective renaturation of denatured and reduced immunoglobulin G In vitro without assistance of chaperone[J].Protein Eng,1996,9:95-100
[80]鲍淑青.重组肌肉生长抑制素的制备及主动免疫生长猪的研究[D].四川:四川农业大学,2007.
[81]Zimmers T A,Davies M V,Koniaris L G,et al.Induction of cachexia in mice by systemically administered myostatin[J].Sciecne,2002,296:1486-1488
[82]马现永,曹永长,舒鼎铭,等.肌肉生成抑制因子基因的克隆、表达及动物免疫实验[J].中国科学C辑 生命科学,2004,34(6):522-526
[83]尹庆宁,孙学权.给仔猪注射猪瘟和口蹄疫疫苗产生不良反应的报告[J].当代畜禽养殖业,2005,(3):9
[84]Price S J,Gresves D R,Watkins H.Identification of novel,functional genetic variants in the human matrix metalloproteinase-2 gene[J].The Journal of Biological Chemistry,2001,276(10):7549-7558.
[85]邵西兵.免疫Leptin对肉种鸡产蛋率和脂肪沉积的影响[D].广州:华南农业大学,2004.
[86]陈丽玲,高士争.重组瘦蛋白对实验性肥胖SD大鼠生长性能影响[J].中国临床医药研究杂志,2007,12:1-2
[87]艾薇,番玮,刘莉.重组瘦蛋白对猪生长性能的影响[J].江西农业学报,2007,19(5):103-104
[2]Carlson C J,Booth F W,Gordon S E.Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading[J].American Journal of Physiology Regulatory Integrative and Comparative Physiology,1999,277:601-606
[3]Kocamis H,Killefer J.Myostatin expression and possible functions in animal muscle growth[J].Domestic Animal Endocrinology,2002,23:447-454.
[4]Roberts S B,Goetz F W.Differential skeletal muscle expression of myostatin across teleost species,and the isolation of multiple myostatin isoforms[J].FEBS Letters,2001,491(3):212-216.
[5]Ji S,Losinski R L,Cornelius S G.Myostatin expression in porcine tissues:tissue specificity and developmental and postnatal regulation.Am J Physiol,1998,275:R1265-R1273.
[6]Xu C,Wu G,Zohar Y,Du S J.Analysis of myostatin gene structure,expression and function in zebrafish[J].The Journal of Experimental Biology,2003,206:4067-4079.
[7]Radaelli G,Rowlerson A,Mascarello F,et al.Myostatin precursor is present in several tissues in teleost fish:a comparative immunolocalization study[J].Cell Tissue Res.,2003,311:239-250
[8]Salamonsen L A,Dimitriadis E,Robb L.Cytokines in implantation[J].Seminars in Reproductive Medicine,2000,18(3):299-310
[9]McPherron A C,Lee S J.Double muscling in cattle due to mutations in the Myostatin gene[J].Proc Natl Acad Sci USA,1997,94:12 457-12 461.
[10]Jeanplong F,Sharma M,Somers W G,etal.Genomic organization and neonatal expression of the bovine myostatin gene[J].Mol Cell Biochem,2001,220(1-2):31
[11]Stratil A,Kopecny M.Genomic organization,sequence and polymorphism of the porcine myostatin(GDF8,MSTN)gene[J].Anim Genet,1999,30(6):468-70.
[12]Sazanov A,Ewald D,Buitkamp J,et al.Amolecular marker for the chicken myostatin gene(GDF8)mapsto 7p11[J].Anim Genet,1999,30(5):388-9.
[13]Thomas M,Langley B,Berry C et al.Myostatin,a negative regulator of muscle growth,functions by inhibiting myoblast proliferation[J].Biol Chem,2000;275:40235-40243
[14]Grobet L.A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[J].Nat Genet,1997,17(1):71-74.
[15]Kambadur R,Sharma M Smith T P L,et al.Mutations in myostatin(GDF-8)in Dubled-muscled Belgian Blue and Piedmontese Cattle[J].PCR Methods Appl,1997,7(9):910-915.
[16]李绍华,熊远著,郑嵘,等.猪MSTN基因多态性及其SNPs的研究[J].遗传学报,2002,29(4):326-331.
[17]Lin J,Heather B.Myostatin knockout in mice increase myogenesis and decreses adipogensis[J].Bioch Biop Res Comm,2002,292(3):701-706.
[18]Jannel Acosta,Yamila Carpio,Ingrid Borroto,et al.Myostatin gene silenced by RNAi show a zebrafish gaint phenotype[J].Journal of Biotechnology,2005,119(4):324-331
[19]McPherron A C,Lee S J.Suppression of body fat accumulation in Myostatin deficient mice[J].The Journal of Clinical Investigation,2002,109(5):595-601.
[20]Kim H S,Liang L,Dean R G,Hausman D B,Hartzell D L,Baile C A.Inhibition of preadipocyte differentiation by myostatin treatment in 3T3-L1 cultures.Biochemical and Biophysical Research Communications,2001,281(4):902-906.
[21]Rebbapragada A,Benchabane H,Wrana J L,Celeste A J,Attisano L.Myostatin signals through a transforming growth factor-like signaling pathway to block adipogenesis.Molecular and Cellular Biology,2003,23(20):7230-7242.
[22]Reardon K A,Davis I.Myostatin,insulin-like growth factor-1,and leulemia inhibitoryfactor mRNAs are upregulated in chronic human disuse muscle atrophy[J].Muscle Nerve,2001,24(7):893-899.
[23]Nester F,Gonzalez-Cadevid,Wayne E,et al.Organization of the human myostatin gene and expression in healthy men of HIV-infected men with muscle wasting[J].Proc Natl Acad Sci USA,1998,95(25):14938-14943.
[24]Gilson H,Schakman O,Combaret L,et al.Myostatin gene deletion prevents glucocorticoid-induced muscle atrophy[J].Endocrinology,2007,148(1):452-460.
[25]Liu W,Thomas SG;Asa SL,Gonzalez-Cadavid N,Bhasin S,and Ezzat S.Myostatin is a skeletal muscle target of growth hormone anabolic action[J].J Clin Endocrinol Metab.2003,88(11):5490-5496.
[26]Mark Thomas,Brett Langley,CaroleBerry,et al.Myostatin,a negativeregulator of muscle growth,functions by inhibiting myoblast proliferation[J].J biol Chem,2000,275(51):40235-40243.
[27]Rios R,Carneiro I,Arce V M,et al.Myostatin is an inhibitor of myogenic differentiation[J].Am J Physiol Cell Physiol,2002,282(5):993-999
[28]Joulia D,Bernardi H,Garandel V,et al.Mechanisms involved in the inhibition of myoblast proliferation and differentiation by Myostatin[J].Exp Cell Res,2003,286(2):263-275
[29]Zhu X,Hadhazy M,Wehling M,et al.Dominant negative myostatin produces hypertrophy without hyperplasia in muscle[J].FEBS Lett,2000,474(1):71-75
[30]Rios R,Carneiro I,Arce VM,etal.Myostatin regulates cell survival during C2C12myogenesis[J].Biochem Biophys Res Commun,2001,280(2):561-566.
[31]Georges M,Grobet L,Pirottin D.Towards transgenic engineering of the myostatin gene in livestock.BiotechNet Proceedings,2004,1-4.
[32]陈晔华,张传茂,陈俭,等.分子细胞生物学[M].北京:清华大学出版社]2006:358-378
[33]Zhang Y,Proenca R,Maffei M,et al.Positional cloning of the mouse obese gene and its human homologue.Nature,1994,372:425-432.
[34]孙长颢,张宏伟,王舒然,等.不同膳食构成对大鼠肥胖基因表达的影响[J].中国公共卫生,1998,14(5):277-280
[35]Ingalls,A.M.,Dickie,M.M.& Snell,G.D.,OBese,a new mutation in the house mouse[J].Hered.1950,41,317-318.
[36]Hummel,K.P.,Dickie,M.M.,and L.,C.D..Diabetes,a new mutation in the mouse[J].Science,1966,153:1127-1128.
[37]Coleman D L.Effects of parabiosis of OBese with diabetes and normal mice.Diabetologia,1973,9:294-298.
[38]Tartaglia L,Dembski M,Wang X,et al.Identification and expression cloning of a leptin receptors,OB-R[J].Cell,1995,83:1263-1271.
[39]Friedman,J.M.,Halaas,J.L.,Leptin and the regulation of body weight in mammals,Nature,1998,395:763-770.
[40]R.S.Ahima,C.Prabakaran,C.Mantzoros,et al.Role of leptin in the neuroendocrine response to fasting[J].Nature.1996,382:250-252.
[41]屈会起,邱明才,林珊.瘦素与肾脏[J].国外医学内科学分册,2000,27(3):111-114
[42]Levin,et al.Decrease food intake does not completely account for adiposity reduction after ob protein infusion[J].Proc Natl Acad Sci USA,1996,93:1726-1730.
[43]Chen,et al.Disappearance of body fat in normal rats induced by adenovirus-mediated liptin gene therapy[J].Proc Natl Acad Sci USA,1996,93:14795-14799
[44]Pelleymounter MA,Cullen MH,Baker MB,et al.Effects of the obese gene product on body weight regulation in ob/ob mice[J].Science,1995,269:540-543.
[45]Vogel G.Leptin:a trigger for puberty.Science,1996,274(5292):1466-1467.
[46]JJ Hwa,AB Fawzi,MP Graziano,et al.Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice[J].Am J Physiol,1997,(8):1204-1209.
[47]Carro E,Pinila L,Seoalle LM,et al.Influence of endogenous leptin tone on the estrous cycle and luteinizing hormone pulsatility in female rats[J]Neuroendocrinology,1997,66:375-377
[48]Spicer L J,Francisco C C,The adipose obese gene product,lepting edidence of a direct inhibitory role in ovarian function[J].Endocrinology,1997,138:3374-3379.
[49]Brogan R S,Mitchel S E,Trayhum P,et al.Suppression of leptin during lacation:contribution of the suckling stimulus versus milk producfion [J].Endocrinology,1999,140(6):2621-2627.
[50]丛琳.肥胖蛋白受体.国外医学卫生学分册,2001,28(4):232-234
[51]吴坚,邹大进.瘦素受体的研究进展.生理科学进展.2000,31(2):143-146
[52]许梓荣,赵国芬,郑家茂.Leptin生物学功能的研究进展及应用前景[J].动物营养学报.2001,13(1):1-5
[53]郭亚宁,候水生,刘小林,等.Leptin的生物学功能研究进展[J].中国畜牧兽医,2002,29(5):26-29.
[54]Yu WH,Kimura M,Walczewska A,et al.Role of leptin in hypothalamic-pituitary function[J].Proc Natl Acad Sci USA,1997,94:1023-1028.
[55]Legradi G,Emerson CH,Ahima RS,et al.Leptin prevents fasting-induced suppression of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypothalamic paraventricular mucleus.Endocrinololgy,1997,138:2569-2576.
[56]兰干球,李芳芳,黄雄军,等.广西巴马小型猪肥胖基因(Leptin)成熟肽序列的cDNA的克隆、序列分析[J]。广西农业生物科学,2007,4
[57]Ma X Y,Cao Y C,Shu D M,et al.Cloning and expression of swine myostatin gene and its application in animal immunization trial.Science in China Ser.C Life Seciences,2005,48(4):368-374.
[58]姜运良,李宁,吴常信,等.不同品种猪肌肉生长抑制素基因单核苷酸多态性分析[J].遗传学报,2001,28(9):840-845
[59]RAMSAY T G,YAN X,MORRISON C,et al.The obesith gene in swine [J].Sci,1998,76(2):484-490.
[60]张冰,杨公社,孙超,等.Leptin基因对陆川猪和大白猪产仔数的影响[J].畜牧兽医学报.2007,38(6):608-613
[61]Lagonigro R,Wiener P,Pilla F,et al.A new mutation in the coding region of the bovine leptin gene associated with feed intake[J].Animal Genetics.2003,34:371-374.
[62]Kimchi-Sarfaty C,Oh J M,Kim I W,et al.A "Silent" polymorphism in the MDR1gene changes substrate specificity[J].Science,2007,315(5811:525-528
[63]Sharma M,Kambadur R,mattews K G,et al.Myostatin,a transforming growth factor-[beta]superfamily member,is expressed in heart muscle and is upregulated in cardiomyocytes after infarct[J].Cell Physiol,1999,180:1-9
[64]徐万祥,邱德义,顾少华,等.去N端信号肽和C端跨膜区猪卵透明带-3B 蛋白在原核系统表达的研究[J].生物工程学报,2001,7(1):16-19
[65]Karl R,Afzalpurkar A,Gupta S K,et al.Expression of bomet monky(Macaca radiata)Zona pellucida-3(zp3)in a prokatyotic system and its immunogenicity.Mol Reprod Dev[J].1997,47:140-147
[66]卢圣栋,《现代分子生物学实验技术》第2版[M],中国协和医科大学出版社,1999
[67]金元昌,刘利,苏晓艳,李景鹏,李会东,周建红IPTG诱导浓度、时间及温度对重组促性腺激素释放激素基因表达的影响[J]黑龙江畜牧兽医.2006,8:17-19
[68]何忠效,张树政.生物化学实验技术丛书—电泳 第2版[M],科学出版社,1999
[69]李春丽,陈其新,何国庆 植物甜蛋白des-pGlul-brazzein在大肠杆菌中表达[J].食品与发酵工业.2006,32(7):8-11
[70]卢圣栋,等.现代分子生物学实验技术第2版[M],中国协和医科大学出版社,1999
[71]蒲勤.人骨形成蛋白2成熟肽在大肠杆菌中的表达、纯化及其生物学活性的研究[M].第四军医大学博士学位论文,2000
[72]戴茹娟.猪肥胖基因(OB)和肥胖基因(OB-R)cDNA的克隆与分析[D].北京:中国农业大学,1998.
[73]J.萨姆布鲁,D.W.拉赛尔,等.《分子克隆实验指南》第三版[M].北京:科学出版社,2002:1682-1683
[74]于自然,黄熙泰,等.《现代生物化学》[M].北京:化学工业出版社,2001.
[75]黎敏义.猪抑制素α亚基重组融合蛋白的制备及应用研究[D].广州:华南农业大学,2006.
[76]许正平,段治军,陈长征,等.NMT在大肠杆菌中的His_6融合表达及其纯化研究.生物化学与生物物理学报.[J]1997,29(4):383-388
[77]陈长征,黄华,龚健,等.人肠杆菌His_6融合表达载体及其表达产物的一步纯化.[J]生物化学与生物物理学报.1996,28(5):523-529
[78]QIAGEN.A handbook for high-level expression and purification of 6×His-tagged proteins.[M]Fifth Edition,2003
[79]Maeda Y,Ueda T,Imoto T.Effective renaturation of denatured and reduced immunoglobulin G In vitro without assistance of chaperone[J].Protein Eng,1996,9:95-100
[80]鲍淑青.重组肌肉生长抑制素的制备及主动免疫生长猪的研究[D].四川:四川农业大学,2007.
[81]Zimmers T A,Davies M V,Koniaris L G,et al.Induction of cachexia in mice by systemically administered myostatin[J].Sciecne,2002,296:1486-1488
[82]马现永,曹永长,舒鼎铭,等.肌肉生成抑制因子基因的克隆、表达及动物免疫实验[J].中国科学C辑 生命科学,2004,34(6):522-526
[83]尹庆宁,孙学权.给仔猪注射猪瘟和口蹄疫疫苗产生不良反应的报告[J].当代畜禽养殖业,2005,(3):9
[84]Price S J,Gresves D R,Watkins H.Identification of novel,functional genetic variants in the human matrix metalloproteinase-2 gene[J].The Journal of Biological Chemistry,2001,276(10):7549-7558.
[85]邵西兵.免疫Leptin对肉种鸡产蛋率和脂肪沉积的影响[D].广州:华南农业大学,2004.
[86]陈丽玲,高士争.重组瘦蛋白对实验性肥胖SD大鼠生长性能影响[J].中国临床医药研究杂志,2007,12:1-2
[87]艾薇,番玮,刘莉.重组瘦蛋白对猪生长性能的影响[J].江西农业学报,2007,19(5):103-104