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目的构建鲍曼不动杆菌外膜蛋白A1S_0115胞外区(A1S_0115A)与GST标签融合表达的原核表达载体,在大肠杆菌XL-1 blue中表达、纯化A1S_0115A蛋白后,进行抗原免疫保护性的初步研究。方法利用生物信息学技术分析A1S_0115蛋白结构,确定蛋白的胞外区片段,PCR扩增该基因片段后,构建重组表达载体p GEX-6P-2-A1S_0115A,将重组载体转化大肠杆菌XL-1blue,IPTG诱导表达GST-A1S_0115A融合蛋白,采用GST亲和层析填料纯化并酶切获得A1S_0115A蛋白。利用已建立的Balb/c小鼠鲍曼不动杆菌全身感染模型对该蛋白抗原的免疫保护性进行初步评价。结果重组质粒经过Bam HⅠ和NotⅠ双酶切鉴定、核酸序列测定和IPTG诱导表达及酶切后蛋白的SDS-PAGE分析表明,A1S_0115A蛋白胞外区相对分子质量大小约50 000,GST标签相对分子质量大小约26 000,与预期设想符合,目的蛋白纯度在95%以上。用A1S_0115A蛋白对小鼠进行2轮次免疫保护性实验及其抗体的被动免疫实验,免疫攻毒后小鼠的保护率分别为50%和55%,被动免疫保护率为45%和50%。结论成功构建重组表达载体p GEX-6P-2-A1S_0115A,利用大肠杆菌可溶表达系统、GST亲和层析和酶切方法获得了高纯度的A1S_0115A蛋白。动物免疫攻毒保护实验结果表明A1S_0115A蛋白具有较好的免疫保护性,为研制新型有效的鲍曼不动杆菌疫苗奠定实验基础。
OBJECTIVE: To construct a prokaryotic expression vector for fusion expression of the extracellular domain A1S_0115 of Acinetobacter baumannii (A1S_0115A) and GST tag in Escherichia coli XL-1 blue and to purify the protein A1S_0115A before immunoprotection . Methods The structure of A1S_0115 protein was analyzed by bioinformatics technology to determine the extracellular region of the protein. After PCR amplification of the gene fragment, the recombinant expression vector pGEX-6P-2-A1S_0115A was constructed. The recombinant vector was transformed into E.coli XL- IPTG induced expression of GST-A1S_0115A fusion protein, purified by GST affinity chromatography and digested to obtain A1S_0115A protein. The immune protection of this protein antigen was preliminarily evaluated using established model of systemic infection in Balb / c mouse Acinetobacter baumannii. Results The recombinant plasmids were identified by restriction endonucleases Bam HI and Not I. The results of nucleic acid sequence analysis and IPTG induction and SDS-PAGE analysis showed that the relative molecular mass of A1S_0115A extracellular domain was about 50,000 and the relative molecular mass of GST tag The size of about 26 000, in line with the expected idea, the purity of the target protein is above 95%. The mice were immunized with A1S_0115A for two times and their passive immunization experiments were performed. The protective rates of mice immunized with A1S_0115A were 50% and 55% respectively, and the passive immunization rates were 45% and 50% respectively. Conclusion The recombinant expression vector pGEX-6P-2-A1S_0115A was successfully constructed. The highly purified A1S_0115A protein was obtained by using E. coli soluble expression system, GST affinity chromatography and enzyme digestion. The experimental results of animal immune challenge protection show that A1S_0115A protein has good immunoprotective properties, which lays the foundation for the development of a new and effective Acinetobacter baumannii vaccine.