抗G17单链抗体-AP融合蛋白的构建、表达与鉴定

Construction,expression and identification of anti-G17 single chain antibodyAP fusion protein

目的:为了便于临床检测,建立一种G17新型免标自指示剂,用原核表达系统表达抗胃泌素单链抗体融合蛋白(ScFv-AP)。方法:通过构建重组质粒pET32a-scFv-AP,选择合适的感受态细胞Rosetta(DE3)及优化表达条件制得目的蛋白。利用固定化金属离子亲和层析技术得到纯品蛋白。再将其作为检测探针运用到双抗体夹心ELISA的方式证明其具有免疫活性与实用性。结果:经Sanger法测序、SDS-PAGE、Western blot证明经金属离子亲和层析技术得到高纯度目的蛋白。经ELISA验证其最低检出限为0.978 ng/ml。实验后确定最佳反应条件为25℃、1 mmol/L IPTG、6 h,每升菌可收获目的蛋白约9.6 mg。目的蛋白具有较高的灵敏度。结论:成功表达具有免疫反应性的抗胃泌素单链抗体融合蛋白。其具有指示蛋白的作用,与传统酶标抗体相比灵敏度高4倍。

Objective:To create a novel label-free self-indicator of G17 for easy clinical detection. Using a prokaryotic expression system to express the anti-gastrin single chain antibody fusion protein(scFv-AP).Methods:The target protein was produced by constructing a recombinant plasmid pET32 a-scFv-AP,selected a suitable sensory cell Rosetta(DE3)and optimized the expression conditions. Immobilized metal ion affinity chromatography was used to obtain pure proteins. The immunoreactivity and practicality were demonstrated by using the pET32 a-scFv-AP plasmid as a detection probe for double antibody sandwich ELISA.Results:Sanger sequencing,SDS-PAGE and Western blot proved that the target protein was obtained by metal ion affinity chromatography with high purity. The optimum reaction conditions were 25℃,1 mmol/L IPTG for 6 h,and 9.6 mg of target protein could be harvested per liter of bacterium.Conclusion:The immunoreactive anti-gastrin single-chain antibody fusion protein is successfully expressed. It is an indicator protein with 4 times higher sensitivity than conventional enzyme-labeled antibodies.