大黄鱼肌肉生长抑制素-1前肽原核表达、多克隆 - 中国细胞生物学学报.pdf

中国细胞生物学学报 Chinese Journal of Cell Biology 2014, 36(10): 1344– 1349 DOI: 10.11844/cj cb.2014.10.0115 研究论文 大黄鱼肌肉生长抑制素-1前肽原核表达、多克隆 抗体制备和功能验证 应 俊 薛良义* (宁波大学海洋学院, 宁波3152 11) 摘要 肌肉生长抑制素(myostatin, MSTN)在动物肌肉的生长和发育过程中起着重要作用。该 研究通过RT-PCR从大黄鱼(Larimichthys crocea)肌肉组织中克隆肌肉生长抑制素-1前肽基因(MSTN- 1pros), 构建了表达大黄鱼生长抑制素前肽的重组质粒, 并在大肠杆菌BL21(DE3) 中进行表达, 获得了 大黄鱼MSTN-1pros融合蛋白。用分离的重组蛋白免疫大白兔, 制备了抗大黄鱼MSTN-1pros的多克 隆抗体。经蛋白免疫印迹法检测, 确定获得的抗体具有较高的特异性。通过对大黄鱼幼鱼的注射实 验, 发现注射过MSTN-1pros抗体的大黄鱼增重率比对照组降低了20.71%(P0.05), 证明MSTN前肽抗 体对大黄鱼的生长具有抑制作用。实验结果表明, MSTN前肽蛋白可以用于养殖鱼类的生长调控。 关键词 肌肉生长抑制素前肽; 大黄鱼; 原核表达; 多克隆抗体; 肌肉生长 Large Yellow Croaker MSTN-1 Prodomain Prokaryotic Expression, Polyclonal Antibody Preparation and Antibody Function Identification Ying Jun, Xue Liangyi* (School of Marine Sciences, Ningbo University, Ningbo 315211, China) Abstract Myostatin (MSTN) plays an important role in the muscle growth and development of animals. MSTN-1 prodomain (MSTN-1pros) gene was cloned from the muscle tissue of large yellow croaker (Larimichthys crocea) by RT-PCR. Recombinant expression vectors containing large yellow croaker MSTN-1pros were constructed and transformed into E. coli BL21 (DE3). The recombinant fusion protein was induced with IPTG and isolated by SDS. The isolated fusion protein was used to raise the polyclonal antibody of MSTN-1pros by immunizing rabbit. The result of Western blot showed that the polyclonal antibody was highly specific. The weight gain rate of the juvenile large yellow croaker injected with MSTN-1pros antibody was 20.71% lower than that of the control group (P0.05). The results indicated that MSTN-1pros antibody had a biological function of inhibiting the muscle growth of lar