农业生物技术学报2012，Vol.20Issue(6)：676-681,6.DOI:10.3969/j.issn.1674-7968.2012.06.012
兔多杀性巴氏杆菌外膜蛋白A(OmpA)重组蛋白单克隆抗体的制备及潜在应用
Preparation and Potential Application of Monoclonal Antibody against Pasteurella multocida Outer Membrane Protein A (OmpA) Recombinant Protein
摘要
Abstract
The aim of this study was to prepare monoclonal antibody (McAb)against Pasteurella multocida. The DNA fragment encoding the mature domain of P. multocida outer membrane protein A (OmpA) was amplified from the genomic DNA and sub-cloned into pET28a (+) expression vector, 37.6 kD rOmpA fusion protein was expressed mainly as an insoluble protein, optimal sohibilization of the recombinant protein was obtained using 8 mol/L urea in lysis buffer. BALB/c mice (Mus musculus) were subcutaneously injected with 100 μg of P. multocida OmpA emulsified by equivolumminal freund's complete adjuvant at the age of 6-8 weeks. Thereafter they were boosted two times with 200 μg of P. multocida OmpA emulsified by Freund's incomplete adjuvant at intervals of three weeks. The spleen cells of BALB/ c mice immunized with recombinant Pm OmpA were collected and infused with SP2/ 0 cell. Sebsequently four hybridoma cell strains were obtained by indirect enzyme linked immunosorbent assay (ELISA). The ELISA titers of antibodies in culture supernatant were 1:128,1:128,1:256 and 1:128, respectively, and ascites titers were 1:6 400,1:6 400,1: 12 800 and 1:6 400, respectively. The McAbs did not cross-react with other gram-negative and gram-positive bacterial pathogens, including E. coli, Bordetella bmnchiseptka, Pseudomonas aeruginosa and slaphylococcus. High titer McAbs were secreted from the hybridoma cells after repeat freezing. The result of Western blotting assay showed that the four Mabs could react with Pm OmpA protein specifically. ELISA test revealed that the 2A2 McAb belonged to the subtype of IgG2b, with a concentration was 130 u-g/mL after protein A affinity purification. The purified 2A2 McAb was selected by Western blot and IFA assays. The result indicated that the McAb could react with the Pm isolate strain. The success of this study has built up a solid base for developing a novel diagnostic methodology to the Pasteurella multocida infection in rabbits.关键词
多杀性巴氏杆菌/外膜蛋白A(OmpA)/重组蛋白/单克隆抗体Key words
Pasteurella multocida/ Outer membrane protein A (OmpA)/ Recombinant protein/ Monoclonal antibody引用本文复制引用
刘燕,庞安娜,韦强,肖琛闻,鲍国连,季权安,钱微..兔多杀性巴氏杆菌外膜蛋白A(OmpA)重组蛋白单克隆抗体的制备及潜在应用[J].农业生物技术学报,2012,20(6):676-681,6.基金项目
国家兔产业技术体系(No.CARS-44-C-2) （No.CARS-44-C-2）
