黄河鲤肌肉发育关键因子的多克隆抗体制备及验证

Preparation and Detection of Polyclonal Antibodies of Key Factors for Muscle Development of Yellow River Carp(Cyprinus carpio haematopterus)

目前,水产养殖动物肌肉发育的研究多集中于转录水平,而蛋白质水平的研究较少,这种局限性制约了对水产养殖动物肌肉发育及生长的深入研究。本研究以黄河鲤白肌为原材料,采用原核表达的技术获得含抗原决定簇的配对盒转录因子7(Pax7)、肌肉转录调节因子(MyoD)、肌细胞生成素(MyoG)和生肌调节因子4(MRF4)4种融合蛋白,经纯化后免疫新西兰大耳兔,获得相应黄河鲤兔源多克隆抗体。采用酶联免疫吸附测定(ELISA)方法检测效价,并以此抗体为工具,随机选取3尾黄河鲤,检测其白肌中Pax7、MyoD、MyoG和MRF4在蛋白质水平的表达情况。结果显示:4种融合蛋白的分子质量分别为15.60、16.90、16.91和19.35 ku,抗体的效价均达到2.4×106;经检测,黄河鲤白肌中Pax7、MyoD、MyoG和MRF4蛋白分子的免疫荧光信号强度无显著差异(P>0.05)。本研究制备的黄河鲤肌肉发育关键因子的多克隆抗体可为进一步定量分析黄河鲤肌肉发育关键标志性蛋白分子的表达提供有力的工具。

Most studies on muscle development of aquaculture animals on transcription level,and few studies on protein level.This limitation restricts in-depth research on muscle development and growth of aquaculture animals.In this study,the white muscle of Yellow River carp(Cyprinus carpio haematopterus)as raw material,the four fusion proteins-paired box 7(Pax7),myogenic differentiation antigen(MyoD),myogenin(MyoG)and myogenic regulatory factor 4(MRF4)for muscle development with antigenic determinant of Yellow River carp were obtained by prokaryotic expression technique.The fusion proteins were purified and used as antigen to immunize the New Zealand rabbits,correspondingly,four Yellow River carp polyclonal antibodies from rabbits were obtained.The enzyme linked immunosorbent assay(ELISA)method was used to evaluate the titers of antiserum,and the antibodies were used to detect the protein expression levels of the Pax7,MyoD,MyoG and MRF4 in white muscle of Yellow River carp(n=3).The results showed that the molecular weight of the four fusion proteins were about 15.60,16.90,16.91 and 19.35 ku,and the titers of the antiserum were all up to 2.4×106.And there were no significant differences in the immunofluorescence signal intensities of Pax7,MyoD,MyoG and MRF4 in the white muscle of Yellow River carp(P>0.05).In conclusion,the polyclonal antibodies of key factors for muscle development prepared in this study can provide a powerful tool for further quantitative analysis of the expression of key marker proteins for muscle development in Yellow River carp.