铜绿假单胞菌快速检测胶体金免疫层析试纸的制备  

Preparation of colloidal gold immunochromatographic test strip for rapid detection of Pseudomonas aeruginosa

作　　者：张世凯 朱辉煌 李家吉 王毅 胡征[1,2,3] ZHANG Shikai;ZHU Huihuang;LI Jiaji;WANG Yi;HU Zheng(School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068,Hubei Province,China)

机构地区：[1]湖北工业大学生物工程与食品学院,湖北武汉430068 [2]湖北工业大学发酵工程教育部重点实验室,湖北武汉430068 [3]工业发酵湖北省协同创新中心,湖北武汉430068

出　　处：《中国生物制品学杂志》2023年第8期941-946,954,共7页Chinese Journal of Biologicals

基　　金：湖北省科技厅自然科学青年基金(2017CFB226)。

摘　　要：目的 研制快速、准确检测铜绿假单胞菌(P.aeruginosa,Pa)的胶体金免疫层析试纸。方法 采用生物信息学对铜绿假单胞菌外膜蛋白OprF进行分析后,化学合成抗原决定簇丰富、种内同源性高的基因序列,连接至pET-28a(+)载体,构建表达载体pET-28a-OprF,转入E.coli BL21(DE3)中,IPTG诱导表达,表达产物经Ni SepharoseTM6 Fast Flow纯化。将纯化的重组OprF蛋白免疫2只雌性BALB/c小鼠,共免疫3~4次,首次免疫为背部皮下多点注射,后续免疫为腹腔注射。通过动物细胞融合技术筛选单克隆抗体,利用单克隆抗体、双抗夹心免疫层析技术制备铜绿假单胞菌快速检测胶体金免疫层析试纸,并评价其特异性、灵敏度及稳定性。结果 获得2株可配对的单克隆抗体Pa-1#和Pa-2#,抗体效价均达到1∶409 600,均可特异性识别OprF。制备的胶体金免疫层析试纸检测灵敏度为1.0×106CFU/mL,与9种常见的呼吸道病原菌无交叉反应,且稳定性较好。结论 制备的胶体金免疫层析试纸可在15 min内快速检测铜绿假单胞菌,特异性强,且具有良好的稳定性。Objective To develop a colloidal gold immunochromatographic test strip for rapid and accurate detection of Pseudomonas aeruginosa(P.aeruginosa,Pa).Methods After bioinformatics analysis of Pa outer membrane protein OprF,the gene sequence with abundant antigenic determinants and high intraspecific homology was chemically synthesized,and then connected to pET-28a(+)vector to construct the expression vector pET-28a-OprF,which was transformed into E.coli BL21(DE3)and induced by IPTG.The recombinant OprF protein was purified by Ni Sepharose~(TM)6 Fast Flow and used to immunize two female BALB/c mice for 3~4 times by multi-point subcutaneous injection in the back at the first immunization and intraperitoneal injection at subsequent immunizations.The monoclonal antibodies were screened by animal cell fusion technique,and the colloidal gold immunochromatographic test strip for rapid detection of Pa was prepared by using monoclonal antibody and double antibody sandwich immunochromatography technique.The specificity,sensitivity and stability of the test strip were evaluated.Results Two monoclonal antibodies,Pa-1#and Pa-2#,were obtained with the titer of 1∶409600,and both of them recognized OprF specifically.The prepared colloidal gold immunochromatographic test strip showed a sensitivity of 1.0×10~6CFU/mL and had no cross reaction with 9 common respiratory pathogens with a good stability.Conclusion The prepared colloidal gold immunochromatographic test strip can detect Pa rapidly within 15 min,with high specificity and good stability.

关 键 词：铜绿假单胞菌 外膜蛋白OprF 单克隆抗体 胶体金免疫层析试纸 

分 类 号：R446.5[医药卫生—诊断学]