抗人LOC339524单克隆抗体的制备和应用

Preparation and application of monoclonal antibody against human LOC339524 protein

目的制备特异性抗人LOC339524的单克隆抗体(m Ab)并进行应用。方法将LOC339524蛋白的非糖基化抗原序列的编码基因进行三段重复表达,合成的基因克隆到质粒p ET28a上。采用大肠杆菌表达系统获得LOC339524重组蛋白,以此作为免疫原免疫BALB/c小鼠,获得m Ab。ELISA测定抗体特异性及效价。Western blot法鉴定LOC339524重组蛋白、免疫组织化学技术分别检测人心肌组织和H9C2细胞中LOC339524蛋白的表达,采用制备的抗体检测不同心脏病患者血清LOC339524水平。结果筛选出2株能稳定分泌抗人LOC339524蛋白m Ab的杂交瘤细胞株,5-D3和4-F8。其中5-D3效价高于4-F8,达2×106。Western blot法、免疫组织化学技术证明5-D3能特异性识别人和大鼠LOC339524蛋白,应用制备的抗体可成功检测不同心脏病患者血清LOC339524的水平。结论成功制备了特异性好、效价高的抗人LOC339524蛋白m Ab。

Objective To prepare and apply monoclonal antibody（ m Ab） against human LOC339524 protein. Methods The non-glycosylated antigenic gene sequence of the LOC339524 protein was expressed in triplicate to enhance immunogenicity.Then this synthetic gene was connected to p ET28 a plasmid. Recombinant LOC339524 protein was obtained by E. coli expression system and was administered intraperitoneally as an immunogen to BALB / c mice to obtain m Ab. The specificity and titer of the m Ab were characterized by ELISA. Recombinant LOC339524 protein was identified through Western blotting.The expression of the LOC339524 protein in human myocardial tissues and H9C2 cells were detected by immunohistochemistry,and its level in the sera of patients with different heart diseases was detected with the antibody.Results We obtained two hybridoma cell lines,5-D3 and 4-F8,secreting specific m Abs against LOC339524 protein. The titer of 5-D3 was up to 2 × 106,higher than the titer of 4-F8. Western blotting and immunohistochemistry demonstrated that 5-D3 could specifically recognize LOC339524 protein of Homo sapiens and Rattus norvegicus. With the antibody we obtained,we successfully detected the serum level of LOC339524 protein in patients with different heart diseases. Conclusion The m Ab against human LOC339524 protein with good specificity and high titer has been successfully prepared.