鳜鱼肌球蛋白重链基因的原核表达及多克隆抗体的制备

Prokaryotic Expression and Preparation of Polyclonal Antibody of Myosin Heavy Chain Genes in Siniperca Chuatsi

肌球蛋白重链(MyHC)是肌肉中的主要结构和功能蛋白,在肌肉收缩过程中起关键作用.根据鳜鱼肌球蛋白重链基因中高亲水性区域DNA设计一对特异性引物,将该基因片段亚克隆到pET-32a质粒,构建重组表达载体并转化入大肠杆菌BL21中.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可大量表达融合蛋白.将经亲和层析纯化后的融合蛋白免疫新西兰大白兔制备多克隆抗体.ELISA和免疫组化测试结果表明:此抗体有较好的特异性和抗原性.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理论基础和技术支持.

Myosin heavy chain（MyHC） is one of the major structural and contracting proteins of muscle.In this study,the MyHC fragment of Siniperca chuatsi was subcloned into plasmid pET-32a to construct a His-tagged recombinant plasmid pET-MyHC.The vector was transformed into E.coli BL21 to express the His-MyHC fusion protein in the bacteria under induction of IPTG.The fusion protein of about 40 000 was expressed and confirmed by SDS-PAGE and Western blot.The 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 polyclonal 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.