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

制备兔多杀性巴氏杆菌(Pasteurella multocida,Pm)外膜蛋白A(OmpA)重组蛋白单抗,为兔多杀性巴氏杆菌病的诊断提供特异性强,灵敏度高的单克隆抗体。本研究选取并扩增Pm外膜蛋白基因OmpA,原核表达获得了的大小为37.6kD的OmpA重组蛋白,以纯化复性的兔多杀性巴氏杆菌OmpA重组蛋白作为免疫原,按常规方法免疫BALB/c小鼠(Mus musculus),取其脾细胞与SP2/0细胞融合,ELISA筛选出了4株分泌Pm OmpA蛋白单克隆抗体的杂交瘤细胞,分别命名为5D2、BC11、2A2和6A4。其中2A2细胞培养液效价为1∶256,5D2、BC11和6A4效价为1∶128;2A2腹水效价为1∶12800,其余3株达1∶6400。杂交瘤细胞经反复冻存、复苏及多次传代,仍能稳定分泌高效价抗体。Western blot显示,4株单克隆抗体均能与Pm OmpA重组蛋白重组发生特异性反应。用ELISA方法鉴定2A2单克隆抗体亚型为IgG2b,Protein A亲和纯化2A2腹水抗体,获得的单抗浓度为130μg/mL。选取纯化的2A2单克隆抗体进行潜在应用研究,Western blot与间接免疫荧光结果显示,单克隆抗体能与兔多杀性巴氏杆菌分离菌株发生特异性反应。表明利用体外重组表达兔多杀性巴氏杆菌OmpA融合蛋白制成的杂交瘤能够稳定分泌效价高、特异性强的单克隆抗体,可用于兔多杀性巴氏杆菌诊断,本研究结果为兔多杀性巴氏杆菌诊断试剂盒的研制提供技术参数。

Preparation and Potential Application of Monoclonal Antibody against Pasteurella multocida Outer Membrane Protein A (OmpA) Recombinant Protein

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 solubilization 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 bronchiseptica,Pseudomonas aeruginosa and staphylococcus.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 μg/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.