芹菜过敏原蛋白Api g1.02的克隆、表达及单抗制备

Gene Cloning,Protein Expression and Monoclonal Antibody Preparation of Celery Allergen Protein Api g1.02

提取芹菜总RNA,反转录PCR(RT-PCR)扩增出过敏原蛋白Apig1.02基因,经PCR、酶切和序列测定后,得到重组质粒pET-32a-Apig1.02。将重组质粒转化到BL21(DE3)pLys感受态细胞中,用1mmol/L异丙基硫代-B-D-半乳糖苷(IPTG)对转化菌进行诱导表达及SDS-PAGE分析后,将表达蛋白提取纯化,制备单克隆抗体并进行Western blot分析。结果表明芹菜过敏原蛋白Apig1.02在体外获得了高效表达,表达融合蛋白分子质量约35ku,诱导6h后表达量最高,占菌体总蛋白的40%左右;制备的单克隆抗体能与表达蛋白发生免疫印迹反应,说明所表达的蛋白具有良好的免疫原性。该研究结果为建立芹菜过敏原蛋白的免疫学检测方法奠定了基础。

RNA was extracted from celery,the celery allergen protein Api g1.02 gene was amplified by RT-PCR and cloned into the pET-32a expression vector.The recombinant plasmid pET-32a-Api g 1.02 was transformed into E coli BL21（DE3） pLys for expression after being identified with PCR,enzyme digestion and sequencing.The transformed bacteria were cultivated and induced with 1 mmol/L isoproyhhio-B-D-galactoside（IPTG）,the bacteria sediment was collected after exposure to IPTG for 2,3,4,5,6,7 h and analyzed by SDS-PAGE.Monoclonal antibody was prepared after the expressed protein being purified and was analysed by western blot.The results showed the celery allergen protein Api g1.02 was expressed efficiently in fusion protein form,the molecular weight of the fusion protein was 35 ku,the expression level of the fusion protein was highest after exposure to IPTG for 6 h and accounted for approximately 40% of the total cellular proteins;The western blot analysis showed the recombinant protein had a good immunogenicity.This result is a better basic for immunological detection research on celery allergen protein.