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目的克隆SD大鼠热休克蛋白HSPB7基因,通过原核表达、纯化获得HSPB7蛋白,对其活性进行初步研究,并制备其多克隆抗体,为进一步研究HSPB7蛋白奠定基础。方法从SD大鼠心脏组织中提取总RNA,经逆转录PCR得到HSPB7基因,亚克隆入pET-43.1a(+)载体中,转化大肠杆菌BL21(DE3)菌株,获得可溶性表达产物。经纯化、质谱鉴定后进行生物学活性研究,并用纯化蛋白免疫新西兰兔,分离血清,Westernblot法测定抗体效价和特异性。结果原核表达载体pET-43.1a-HSPB7成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相对分子质量(Mr)约18600的HSPB7蛋白,纯化蛋白经质谱鉴定正确。HSPB7具有分子伴侣活性,这种活性具有温度依赖性。用纯化的蛋白免疫新西兰大耳白兔,制备的多克隆抗体效价达1∶16000,且具有较强免疫特异性。结论得到具有生物学活性的大鼠HSPB7蛋白,成功制备了兔抗大鼠HSPB7蛋白的多克隆抗体,为后续研究提供了重要的实验材料。
Objective To clone the heat shock protein HSPB7 gene of SD rat and obtain the HSPB7 protein by prokaryotic expression. The activity of HSPB7 protein was preliminarily studied and its polyclonal antibody was prepared, which laid the foundation for further study of HSPB7 protein. Methods Total RNA was extracted from the heart tissue of SD rats. The HSPB7 gene was obtained by reverse transcription PCR and subcloned into pET-43.1a (+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) to obtain the soluble expression product. After purification and mass spectrometry identification, the biological activity was studied. The purified protein was used to immunize New Zealand rabbits and the serum was separated. The antibody titer and specificity were determined by Western blotting. Results The prokaryotic expression vector pET-43.1a-HSPB7 was successfully constructed and was induced in E. coli BL21 (DE3). HSPB7 protein with a molecular weight of about 18600 was obtained. The purified protein was identified by mass spectrometry. HSPB7 has chaperone activity and this activity is temperature-dependent. Immunized New Zealand white rabbits with the purified protein, the prepared polyclonal antibody titer reached 1: 16000, and has strong immunospecificity. Conclusion The rat HSPB7 protein with biological activity was obtained and the polyclonal antibody of rabbit anti-rat HSPB7 protein was successfully prepared, which provided an important experimental material for the follow-up study.