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目的:在大肠杆菌中表达ESP30蛋白,并制备兔抗ESP30抗体。方法:从嗜水气单胞菌基因组中扩增ESP30基因序列,并克隆入非融合表达载体pDH2中,在温度诱导下,表达目的蛋白,以表达的ESP30作为免疫原免疫家兔制备抗ESP30抗血清,抗体效价及特异性分别用ELISA和Westernblot法鉴定。结果:在大肠杆菌中高效表达相对分子质量(Mr)约为66000的ESP30蛋白。ELISA法检测抗血清的效价可达到1∶128000,Westernblot分析抗血清可与原核表达的ESP30蛋白特异结合。结论:成功地制备兔抗ESP30的抗血清,为进一步研究ESP30蛋白的结构和功能奠定了基础。
OBJECTIVE: To express ESP30 protein in E. coli and prepare rabbit anti-ESP30 antibody. Methods: The ESP30 gene was amplified from the Aeromonas hydrophila genome and cloned into the non-fusion expression vector pDH2. The recombinant protein was expressed under the induction of temperature. The expressed ESP30 protein was used as an immunogen to immunize rabbits to prepare anti-ESP30 antibody Serum, antibody titer and specificity were identified by ELISA and Western blot. Results: ESP30 protein with a relative molecular mass (Mr) of about 66000 was efficiently expressed in E. coli. The titer of antiserum detected by ELISA was up to 1:128000, and the antiserum analyzed by Western blot could specifically bind to prokaryotic expressed ESP30 protein. Conclusion: The antiserum of rabbit anti-ESP30 was successfully prepared, which laid the foundation for further study on the structure and function of ESP30 protein.