张宁宁,白俊杰,何小平,郭玉函,叶星,李胜杰,杜晓东

• 1:广东海洋大学水产学院
• 2:中国水产科学研究院珠江水产研究所中国水产科学研究院热带亚热带鱼类选育与养殖重点开放实验室

摘要(Abstract)：

将大口黑鲈(Micropterus salmoide)肌肉生长抑制素(Myostatin,MSTN)前肽(MSTN-Pro)的cDNA定向克隆到真核表达载体pcDNA3.1(-)/mycHisB中,双酶切检测和测序鉴定证实,插入pcDNA3.1(-)/mycHisB载体中的片段为目的基因的核苷酸序列,MSTN基因前肽cDNA为正向插入,且重组质粒无错配或插入移位等突变。采用肌肉注射法将重组表达质粒注入大口黑鲈背部肌肉组织,在注射后第2天经RT-PCR检测到MSTN前肽基因mRNA的表达,第6天经免疫组化学检测到MSTN前肽蛋白的表达,第8天蛋白表达强度增强,对照组始终未检测到MSTN前肽基因的表达。

关键词(KeyWords)： 大口黑鲈;肌肉生长抑制素(MSTN);真核表达;表达载体;肌肉注射;免疫组织化学

Abstract:

Keywords:

基金项目(Foundation): 国家科技支撑项目(2006BAD01A1209);; 国家科技基础条件平台工作项目(2005DKA21103);; 广东省自然科学基金项目(7301732);; 中国水产科学研究院水产种质资源与养殖技术重点开放实验室开放基金项目

作者(Author): 张宁宁,白俊杰,何小平,郭玉函,叶星,李胜杰,杜晓东

参考文献(References)：

• [1]Mcpheron A C,Lawler A M,Lee S J.Regulation of skeletal muscle mass in mice by a new TGF-βsuperfamily member[J].Nature,1997,387(6628):83-90.
• [2]Grobet L,Martin L J,Poncelet D,et a1.A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[J].Nat Genet,1997,17:71-74.
• [3]Justin P,John S,Daniel B.Making sense of latent TGFβactivation[J].Journal of Cell Science,2003,116:217-224.
• [4]Yang Jinzeng,Zhao Baoping.Postnatal expression of myostatin propeptide cDNA maintained high muscle growth and normal adipose tissue mass in transgenic mice fed a high-fat diet[J].Mol Reprod Dev,2006,73:462-469.
• [5]John S,John G,Daniel B.TGF-βlatency:biological significance and mechanisms of activation[J].Stem cells,1997,15:190-197.
• [6]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.
• [7]Yang J,Ratovitski T,Brady J P,et a1.Expression of myostatin prodomain results in muscular transgenic mice[J].Mol Reprod Dev,2001,60:351-361.
• [8]Amali A A,Lin C J,Chen Y H,et al.Up-regulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish(Danio rerio)by knock-down of myostatin[J].Dev Dyn,2004,229:847-856.
• [9]Rescan P Y,Jutel I,Ralliere C.Two myostatin genes are differentially expressed in myotomal muscles of the trout(Oncorhynchus mykiss)[J].J Exp Bio1,2001,204(Pt 20):3523-3529.
• [10]李胜杰,白俊杰,叶星,等.加州鲈肌肉生长抑制素(MSTN)cDNA的克隆和序析[J].海洋渔业,2007,29(1):13-19.
• [11]赵浩斌,彭扣,王玉凤,等.鱼类肌肉生长抑制素研究进展[J].水生生物学报,2006,30(2):227-231.
• [12]Xu C,Wu G,Zohar Y,et a1.Analysis of myostatin gene structure,expression and function in zebrafish[J].J Exp Bio1,2003,206:4067-4079.
• [13]Wells D J.Gene transfer by intramuscular injection of plasmid DNA[M]//Dickson G.Molecular and cell biology of human gene therapeutics.London:Chapman&Hall,1995:83-103.
• [14]郭玉函.大口黑鲈Myf5基因结构分析和对鱼类肌肉生长作用的研究[D].上海:上海海洋大学,2008.
• [15]茅卫锋,汪亚平,孙永华.虹鳟鱼组蛋白H3启动子的分子克隆及在稀有鮈鲫中表达活性分析[J].自然科学进展,2004,14(1):46-50.
• [16]Gomez-Chiarri M,Chiaverini L A.Evaluation of eukaryotic promoters for the construction of DNA vaccines for aquaculture[J].Genet Anal,1999,15:121-124.
• [17]石军,陈安国,洪奇华.DNA疫苗在鱼类中的应用研究进展[J].中国兽药杂志,2002,36(5):41-45.
• [18]Kanellos T,Sylvester I D,Butler V L,et al.Mammalian granulocyte-macrophage Clony-stimulating factor and some CpG motifs have an effect in the immunogenicity of DNA and subunit vaccines in fish[J].Immunology,1999,96(4):507-510.
• [19]Roorda B D,Hesselink M K,Schaart G,et al.DGAT1 overexpression in muscle by in vivo DNA electroporation increases intramyocellular lipid content[J].Lipid Res,2005,46(2):230-236.
• [20]王留义,孙威,陈明哲,等.人前胰岛素原基因裸质粒DNA肌肉注射可显著降低链脲佐菌素诱发糖尿病小鼠的血糖水平[J].生理学报,2003,55(6):641-647.
• [21]Wolff I A,Malone R W,Williams P,et al.Direct gene transfer into mouse muscle invo[J].Science,1999,247:145-146.
• [22]Danko I,Wolff J A.Direct gene transfer into muscle[J].Vaccine,1994,12(16):1499-1502.
• [23]Maeda S,Ohmori K,Kurata K,et a1.Expression of LacZ gene in canine muscle by intramuscular-inoculation of a plasmid DNA[J].J Vet Med Sci,2004,66(3):337-339.
• [24]鄢庆枇,苏永全,王军,等.绿色荧光蛋白基因在大黄鱼体内的表达[J].厦门大学学报,2003,41(2):265-268.