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目的:制备心肌锚定重复序列蛋白(CARP)的抗体并分析其表达模式。方法:通过生物信息学对其抗原性进行分析,通过RT-PCR从小鼠的心肌cDNA中扩增了N-端279bp的片段,并克隆到表达载体pET28b中,使其在大肠杆菌BL21(DE3)中表达,用镍离子螯合柱(Ni-NTA)纯化CARP蛋白,用纯化的蛋白免疫小鼠制备多克隆抗体。利用制备的抗体,通过Western blot和免疫荧光的方法,分别检测了CARP在各组织和细胞中的表达模式。结果:制备了CARP抗体,进一步验证了CARP表达在蛋白水上呈心肌特异性,在大鼠心肌原代细胞中主要在细胞核中表达。结论:成功制备了CARP抗体,分析了CARP的表达模式,为进一步CARP的功能奠定了基础。
OBJECTIVE: To prepare and analyze the expression pattern of cardiomyocyte anchorage repeat protein (CARP). METHODS: The antigenicity was analyzed by bioinformatics. A 279bp N-terminal fragment was amplified from mouse cardiac cDNA by RT-PCR and cloned into the expression vector pET28b. The amplified fragment was cloned into E. coli BL21 (DE3) The purified CARP protein was purified by Ni-NTA and immunized with the purified protein to prepare polyclonal antibody. The prepared antibody was used to detect the expression pattern of CARP in various tissues and cells by Western blot and immunofluorescence. RESULTS: The CARP antibody was prepared and further verified that CARP expression was cardiac-specific in protein water and mainly expressed in the nucleus in primary rat cardiomyocytes. CONCLUSION: The CARP antibody was successfully prepared and the expression pattern of CARP was analyzed, which laid the foundation for the further CARP function.