【摘要】 目的纯化牙龈卟啉单胞菌牙龈蛋白酶K催化结构域(KGPcd)融合蛋白并制备其多克隆抗体,为下一步的实验提供条件。方法 KGPcd蛋白与载体pET-16b中的His标签融合表达,用Ni-NTA亲和层析纯化融合蛋白,复性后用Western blot检测。以重组、纯化的KGPcd蛋白为抗原,免疫新西兰大白兔,制备多克隆抗体,用间接ELASA法检测抗体效价,Western Blot进行抗血清特异性试验。结果经亲和层析、复性得到纯化融合蛋白His-KGPcd,Western blot进一步证实纯化的蛋白为His-KGPcd融合蛋白。抗血清稀释达1:3200时,ELASA法仍有明显的抗原抗体反应,Western blot也进一步证实抗血清能够与KGPcd发生特异性反应,抗体仅特异识别目的蛋白。结论成功制备兔抗KGPcd多克隆抗体,为后续研究奠定基础。更多还原

【Abstract】 Objective To purify the fusion protein His-KGPcd(gingipain K catalytic domain) from Porphyromonas gingivalis and prepare the polyclonal anti-KGPcd antibody.Methods The gene fragment encoding kgpcd was inserted into prokaryotic expression vector pET-16b.The expressed fusion protein was purified by Ni-NTA affinity chromatography and then renatured by dilution and dialysis.Then the fusion protein was characterized by Western blot.The recombined and purified KGPcd protein combined with the complete Freund′s adjuvant was used to immunize the rabbits.Then the anti-KGPcd antibody was prepared.The antisera were identified by indirect ELISA.Results The fusion protein His-KGPcd was gotten through purification by Ni-NTA affinity chromatography.The antisera could be combined with the KGPcd specifically.Conclusion The polyclonal anti-KGPcd antibody was successfully prepared in this study.更多还原