【摘要】 为深入了解绵羊myostatin基因的表达及调控机理,进行了绵羊myostatin蛋白多克隆抗体的制备。首先将绵羊myostatin基因C-端克隆入含组氨酸标签的表达载体pET-30a-c(+)中,转化大肠杆菌BL21后IPTG诱导表达,然后利用Ni2+亲和层析纯化重组蛋白,薄层扫描及Bradford法分别检测纯化后蛋白的纯度与含量。应用重组蛋白免疫家兔制备多克隆抗体,利用间接ELISA法检测多克隆抗体效价,用Western blot及免疫细胞化学染色检测抗体特异性。结果,薄层扫描分析纯化后蛋白纯度可达95%以上,Bradford法检测蛋白浓度约5 mg.mL-1,制备的抗血清效价可达1∶250 000以上,Western blot检测证明抗体特异性良好,免疫细胞化学染色表明抗血清可检测到肌肉细胞中内源性myostatin的表达,说明所制备的多克隆抗体可以应用于进一步研究,有助于阐明绵羊myostatin的表达及调控机理。更多还原

【Abstract】 To further understand the biological functions of myostatin in sheep and its molecular mechanism,both of the recombinant myostatin protein and its polyclonal antibody are indispensable.The C-domain of sheep myostatin gene was amplified and cloned into the expression vector pET-30a-c(+).After induced by IPTG,sheep myostatin was expressed in E.coli.The recombinant protein was further purified by Ni2+ affinity chromatography and its purity and content were detected by thin-layer scan analysis and Bradford assay,respectively.Thin-layer scan showed that the purity of recombinant protein reached at least 95%.Bradford assay showed the content was about 5 mg·mL-1.Rabbits were immunolized with purified recombinant myostatin.The titer of polyclonal antibody was detected by indirect ELISA assay.The specificity of polyclonal antibody was evaluated not only by Western blot but also Cytochemical stain.The titer of antiserum was as high as 1:250 000,with very high specificity proved by Western blot and Cytochemical stain.The expression of endogenous myostatin was detected in myoblast by the polyclonal antibody.These results showed that recombinant protein of sheep myostatin C-domain with high purity and its specific polyclonal antibody were obtained,and this polyclonal antibody could be used in future research work.更多还原