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目的:克隆、表达藤黄微球菌Rpf结构域多肽,并制备针对该多肽的单克隆抗体(mAb)。方法:采用聚合酶链反应(PCR)藤黄微球菌基因组中扩增出藤黄微球菌Rpf结构域基因,用限制性内切酶消化后插入到pUC-19克隆载体中,测序正确后,再亚克隆到融合表达载体pPro-EXHT中,转化大肠杆菌DH5α,目的基因经IPTG诱导,由T7启动子调控表达了氨基端带6个连续组氨酸残基的Rpf结构域基因多肽,在变性条件下纯化目的蛋白。用纯化的目的蛋白作为免疫原免疫BALB/c小鼠,进行细胞融合、克隆化制备抗Rpf domain单抗(mAb),用ELISA法初步鉴定其特异性和相对亲和力。结果:在变性条件下用Ni2+-NTA亲和色谱柱纯化目的融合蛋白,纯化获得的蛋白纯度大于90%。用该融合蛋白免疫小鼠后,获得了3株抗Rpfdomain mAb,这3株mAb均特异性识别Rpf domain多肽。结论:所获得的抗Rpfdomain mAb特异性强、效价高,对进一步研究Rpf domain在结核病免疫预防中的作用提供了有力的工具。
OBJECTIVE: To clone, express Micrococcus luteus Rpf domain polypeptide and prepare monoclonal antibody (mAb) against the polypeptide. Methods: The gene of Micrococcus luteus was amplified by polymerase chain reaction (PCR) from Micrococcus luteus and inserted into pUC-19 cloning vector after digestion with restriction endonuclease. After sequencing correctly, Was subcloned into fusion expression vector pPro-EXHT and transformed into E. coli DH5α. The target gene was induced by IPTG, and the Rpf domain polypeptide with 6 consecutive histidine residues at the amino-terminal was regulated by the T7 promoter. Purification of the target protein. BALB / c mice were immunized with the purified protein as an immunogen. The fusion protein was cloned and the anti-Rpf domain mAb was cloned. The specificity and relative affinity of the anti-Rpf domain mAb were determined by ELISA. Results: The target fusion protein was purified by Ni2 + -NTA affinity chromatography under denaturing conditions. The purity of the purified protein was over 90%. After the mice were immunized with the fusion protein, three anti-Rpfdomain mAbs were obtained, and all three mAbs specifically recognize the Rpf domain polypeptide. CONCLUSION: The anti-Rpfdomain mAb obtained has high specificity and high titer, which provides a powerful tool for further studying the role of Rpf domain in tuberculosis immunopathology.