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目的制备高效的福氏志贺菌5a M90T3-磷酸甘油醛脱氢酶(GAPDH)的多克隆抗体,并对其特异性进行评价。方法提取M90T的全基因组,采用PCR法扩增M90T中的基因gapA克隆到原核表达载体pET-24中,重组质粒转入到大肠杆菌BL21(DE3)中进行诱导表达,条件优化后纯化GAPDH-His融合蛋白。以纯化的融合蛋白作为抗原免疫BALB/c小鼠,制备该蛋白的多克隆抗体。Western blot法、ELISA鉴定获得的抗血清。结果成功构建了原核表达载体pET24a-gapA,通过一系列诱导条件的优化,确定可溶性条件为30℃、1 mmol/L IPTG诱导2 h。用纯化的融合蛋白GAPDH-His作为抗原免疫小鼠制备多克隆抗体,Western blot法、ELISA证明多克隆抗体制备成功。结论成功制备了福氏志贺菌5a M90T特异性的小鼠多克隆抗体。
Objective To prepare a polyclonal antibody against 5 M90T3-glyceraldehyde-6-phosphate dehydrogenase (GAPDH) from Shigella flexneri and to evaluate its specificity. Methods The whole genome of M90T was extracted. The gene gapA in M90T was amplified by PCR and cloned into prokaryotic expression vector pET-24. The recombinant plasmid was transformed into E. coli BL21 (DE3) for expression. After optimization, GAPDH-His Fusion protein. BALB / c mice were immunized with the purified fusion protein as an antigen to prepare polyclonal antibodies to the protein. Western blot, ELISA to identify the antiserum. Results The prokaryotic expression vector pET24a-gapA was successfully constructed. After optimization of a series of inducing conditions, the soluble condition was determined at 30 ℃ and induced by 1 mmol / L IPTG for 2 h. The purified fusion protein GAPDH-His was used as antigen to immunize mice to prepare polyclonal antibody. Western blot and ELISA showed that polyclonal antibody was successfully prepared. Conclusion Murine Shigella flexneri 5a M90T specific polyclonal antibody was successfully prepared.