论文部分内容阅读
目的采用GST-myostatin融合蛋白进行多克隆抗体的制备、鉴定并进一步阐明神经损伤后腓肠肌肌萎缩过程中myostatin蛋白表达变化。方法将纯化好的GST-myostatin融合蛋白免疫家兔,获得抗血清,经Western blot、免疫荧光组织化学及ELISA检测抗体特异性及效价;建立大鼠胫神经夹伤模型,采用western blot及免疫组织化学法检测胫神经夹伤后不同时间段腓肠肌中myostatin蛋白表达变化。结果得到较高纯度的GST-myostatin融合蛋白,获得兔源的抗myostatin血清,经western blot、免疫组织化学及ELISA实验证实所得抗体具有较高的特异性及效价;证实在胫神经夹伤后,腓肠肌myostatin蛋白水平迅速上升,在第14天达到高峰,随后逐渐下降,至第28天降至接近正常水平。结论成功制备了兔抗大鼠myostatin抗血清,进一步检测表明了胫神经损伤后腓肠肌中myostatin蛋白的表达具有时间依赖性。本研究为阐明myostatin在骨骼肌萎缩过程中的作用提供了实验依据。
Objective To prepare polyclonal antibody using GST-myostatin fusion protein and to identify and further clarify the expression of myostatin in the process of muscular atrophy after gastrocnemius injury. Methods The purified GST-myostatin fusion protein was immunized rabbits to obtain antiserum. Western blot, immunofluorescence histochemistry and ELISA were used to detect antibody specificity and titer. The model of tibial nerve injury in rats was established, and western blot and immunization Changes of myostatin in gastrocnemius muscle after tibial nerve injury at different time points were detected by histochemistry. Results The higher purified GST-myostatin fusion protein was obtained and the rabbit anti-myostatin serum was obtained. The results of western blot, immunohistochemistry and ELISA showed that the obtained antibody had high specificity and titer. , The level of myostatin protein in gastrocnemius muscle increased rapidly and peaked on the 14th day, then decreased gradually to the normal level on the 28th day. Conclusion Rabbit anti-rat myostatin antiserum was successfully prepared. Further tests showed that time-dependent expression of myostatin protein in gastrocnemius muscle after tibial nerve injury. This study provides an experimental basis for elucidating the role of myostatin in skeletal muscle atrophy.