鳜鱼肌球蛋白重链基因(MyHC)原核表达及其多克隆抗体制备-动物遗传育种与繁殖专业论文.docxVIP

摘要 摘要 IIII I I II II II I I I III I IIl Y1 91 9942 摘要 肌球蛋白重链(MyHC)是肌肉的一个主要结构和功能蛋白。本研究根据鳜囱__MyHC 基因亲水性高的区域序列，设计一对特异性引物，将鳜鱼肌球蛋白重链基因片段亚克 隆到pET-32a质粒，构建重组表达载体并转化入大肠杆菌Escherichia coli BL21中。 SDS和Western杂交检测表明，在IPTG诱导下含有重组载体的菌株可表达分子质 量约40 KD的融合蛋白。诱导条件优化实验结果显示，该菌株经0．8 mmol／L IPTG诱 导3 h可大量表达此蛋白。一步亲和层析纯化后，将纯化后的蛋白注射到新西兰大白兔 中制备多克隆抗体。通过ELISA测试抗体，结果表明此抗体有较好的特异性。鳜鱼 MyHC基因表达的研究为深入研究肌球蛋白重链的功能特性及其对肉质性状的影响奠 定基础，为提高鱼类肉质提供重要的理论基础和技术支持。 关键词：鳜鱼；肌球蛋白重链；快速骨骼肌；基因表达 Expression Expression and purification of the myosin heavy chain(MyHC) from the fast skeletal muscle of the Siniperca chuatsi and preparation of its polyclonal antibody Abstract Myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle．In this study,the MyHC fragment of Sinil：Ierca chuatsi was subcloned into plasmid pET-32a to construct a His—tagged recombinant plasmid pET-MyHC．m vector was transformed into E．coli BL2 1 to express the His—MyHC fusion protein in the bacteria under induction of IPTCt The fusion protein of about 40 kDa Was expressed and confirmed by SDSand Western blot．nle concentration of the IPTG and the length of inducing time that affected the protein expression level were optimized．It Was found that the protein Can be expressed at high level after being induced by 0．8 mmol／L IPTG for 3 h．After purification by one-step affinity chromatography,the fusion protein Was injected into New Zealand rabbits to prepare polyelonal antibody．Then the antibody Was tested by ELISA， and the results showed that the antibody had fine specificity．This work on the MyHC gene from the Siniperca chuatsi added useful information for fish molecular biology and fish genomics． KEY WORDS：Siniperca chuatsi；myosin heavy chain：fast skeletal muscle：gene expression Ⅱ 一引言一引 一引言 一引 言 鳜鱼(Siniperca chuatsi)属鲈形目，始科，鳜亚科，鳜属。俗称：鳜花鱼、季花 鱼、桂花鱼、桂鱼。鳜鱼广泛分布于中国东部平原的江河湖泊。鳜鱼肉洁白、细嫩而 鲜美，无小刺，富含蛋白质。每百克可食部分含蛋白质15．5．19．3克，脂肪0．4．3．5克， 热量78．109千卡，钙79．206毫克，磷107．143毫克，铁O．7．5．6毫克，硫胺素O．01毫克， 核黄素0．10毫克，尼克酸1．9毫克。鳜鱼自古就被列为名贵鱼类之一。1972年出土的马 王堆