【摘要】 目的通过对串珠素核心蛋白多克隆抗体的研究,探讨串珠素在咀嚼肌中的表达特点。方法将串珠素片段插入到pET32a表达载体中,经EcoR I+Sal I酶切鉴定后将正确的质粒转化DH5α大肠杆菌,挑克隆后接种于新鲜LB中。对数培养后经聚丙烯酰胺凝胶电泳及蛋白免疫印迹技术检测融合蛋白的表达,并通过免疫组化检测串珠素在咀嚼肌中的表达。结果聚丙烯酰胺凝胶电泳结果显示在IPTG诱导组出现明显的蛋白表达,而未经IPTG诱导的大肠杆菌及pET32a空载体均未见表达条带。蛋白免疫印迹结果亦显示插入pET32a载体的序列表达成功。免疫组化示串珠素在咀嚼肌中有显著表达,在肌膜处呈带状聚集。结论分子克隆技术可以对串珠素进行扩增,不同品系的串珠素蛋白间有交叉免疫原性,串珠素在咀嚼肌肌纤维中有广泛分布。更多还原

【Abstract】 Objective To investigate the expression features of perlecan in masticatory muscles by studying the specific anti core polyclonal antibodies of perlecan.Methods Perlecan fragment was inserted into the pET32a expression vector,and the plasmid identifeied by the EcoR I + of Sal I restriction enzyme digestion was transferred into E.Coli DH5α,the clones were picked out and inoculated in fresh LB.The Expression of fusion protein was detected by polyacrylamide gel electrophoresis and Western Blot,and the expression of perlecan in masticatory muscles was detected by immunohistochemical method.Results Polyacrylamide gel electrophoresis showed that the fusion proteins were found in IPTG induced group,while no nosignals were found in group without IPTG induction and pET32a empty vector.Western Blot also found positive result,immunohistochemical results showed that perlecan in the masticatory muscles was significantly expressed in the sarcolemma and formed zonal aggregation.Conclusions The perlecan can be amplificated by cloning technology,perlecan from different strains of mammals has crossing immunogen,and perlecan widely distributs in masticatory muscles.更多还原