铜绿假单胞菌VgrG1a蛋白的原核表达及其人鼠嵌合型单抗的制备与鉴定
Prokaryotic expression and identification of human-mouse chimeric monoclonal antibody against Pseudomonas aeruginosa VgrG1a protein
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摘要:目的 探究铜绿假单胞菌Ⅵ型分泌系统缬氨酸甘氨酸重复蛋白G1a (VgrG1a)的原核表达方法, 并制备其鼠源性和人鼠嵌合型单克隆抗体, 为抗铜绿假单胞菌感染的诊断和治疗提供基础。方法 根据NCBI网站上查找到的VgrG1a序列构建重组质粒pET-21a-VgrG1a, 转化大肠杆菌感受态细胞BL21(DE3) plysS进行诱导。通过免疫沉淀法和酶联免疫吸附实验(ELISA)对诱导得到的重组蛋白进行表达、纯化和鉴定。用纯化后的VgrG1a重组蛋白免疫BALB/c小鼠, 制备鼠源多克隆抗体, 并利用ELISA方法测定小鼠血清抗体的效价和特异性。通过P3X63Ag8.653骨髓瘤细胞融合小鼠脾细胞制备杂交瘤细胞, 再通过有限稀释法获得单克隆细胞。经多次ELISA筛选出能够稳定表达特异性抗体的细胞株。测序后获得单克隆抗体的序列信息, 并构建人鼠嵌合基因, 设计出含有此嵌合基因抗体的质粒, 转染Expi293细胞, 制备人源化单克隆抗体。结果 实验成功构建了VgrG1a重组质粒, 经探索在最佳表达诱导条件下16℃下使用0.5 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导16 h, 表达得到了重组蛋白VgrG1a。SDS-PAGE凝胶电泳结果显示, 相对分子质量72 000处重组蛋白浓度最高。检测制备的鼠源抗体在稀释到1/640 000后效价仍较高, 并且与目的蛋白能特异性地识别并结合。人鼠嵌合型单克隆抗体8A4F5对抗原VgrG1a亲和力高于鼠源单克隆抗体。结论 该原核表达的重组蛋白具有良好的免疫原性, 利用表达的重组蛋白制备的单克隆抗体具有较高的抗体效价和特异性, 为进一步研究蛋白的结构与功能、研制相关的诊断试剂及疫苗提供了生物材料。Abstract:Objective To explore the prokaryotic expression method of VgrG1a protein in type Ⅵ secretion system of Pseudomonas aeruginosa, and prepare murine and human-mouse chimeric monoclonal antibodies.Methods Sequences of the VgrG1a genes were retrieved from Genbank in National Center for Biotechnology Information (NCBI) website to generate recombinant pET-21a-VgrG1a plasmid.The plasmid was transformed to E.coli competent cells BL21(DE3) plysS for induction.The induced recombinant protein was purified and identified by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA).BALB/c mice were immunized with purified VgrG1a recombinant protein, and mouse polyclonal antibodies were prepared.The titer and specificity of mouse serum antibodies were determined by ELISA.Hybridoma cells were fused of mouse B-cells with myeloma tumour cells (P3X63Ag8.653).Then monoclonal cells were obtained by limited dilution method.Stable cell lines were selected harboring and expressing specific antibodies screened by ELISA.Gene of the antibody was acquired by sequencing.The plasmid of human-mouse chimeric gene was transfected into Expi293 cells to prepare human monoclonal antibodies.Results The recombinant plasmid of VgrG1a was successfully constructed.Next, the VgrG1a protein was over expressed and purified in E.coli, and the best induction conditions were 16 h at 16℃ with 0.5 mmol/L IPTG.The results of SDS-PAGE gel electrophoresis showed that the recombinant protein concentration was the highest at the relative molecular mass of 72 000.The titer of the prepared mouse antibody was still high after diluted to 1/640 000 with specifically recognition and binding to the target protein.The affinity of human-mouse chimeric monoclonal antibody 8A4F5 against antigen VgrG1a was higher than that of mouse monoclonal antibody.Conclusions The recombinant protein has good immunogenicity, and the monoclonal antibody prepared by recombinant protein has high antibody titer and specificity, which provides biomaterials for further study of protein structure and function, contributing to the development of diagnostic reagents and vaccines.
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