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目的验证免疫蛋白质组中鉴定到的S-腺苷甲硫氨酸MetK蛋白的免疫原性。方法免疫蛋白质组学的方法鉴定到MetK蛋白,通过PCR方法扩增metK基因全长,然后将其克隆到原核表达载体pET32a中,转化大肠杆菌DH5α,测序正确后提取质粒转入BL21(DE3)中进行诱导表达。再通过Ni-NTA柱纯化MetK蛋白,纯化后的蛋白免疫SPF级的BALB/c小鼠,制备多克隆抗体,通过多克隆抗体与炭疽芽孢杆菌A16R全菌蛋白Western印迹检测,验证MetK蛋白的免疫原性。结果与结论成功表达了MetK蛋白,制备了其多克隆抗体,为免疫蛋白质组学鉴定到的蛋白点进行验证,也为研究MetK蛋白的生物学功能研究提供了条件。
Objective To verify the immunogenicity of the S-adenosylmethionine MetK protein identified in the immunoproteome. Methods MetK protein was identified by immuno proteomics. The full-length metK gene was amplified by PCR and cloned into prokaryotic expression vector pET32a. The recombinant plasmid was transformed into E. coli DH5α. After sequencing, the plasmid was transformed into BL21 (DE3) Induction of expression. MetK protein was purified by Ni-NTA column, and the purified protein was immunized with BALB / c mice of SPF grade to prepare polyclonal antibody. The immunization of MetK protein was confirmed by Western blotting of polyclonal antibody and whole-cell protein of Bacillus anthracis A16R Original. RESULTS AND CONCLUSION MetK protein was successfully expressed and its polyclonal antibody was prepared. The protein spots identified by immuno proteomics were validated, which also provided the conditions for studying the biological function of MetK protein.