赭曲霉毒素A单克隆抗体的体外制备及其重组抗体的构建研究

目的 实现抗赭曲霉毒素A(ochratoxinA,OTA)单克隆抗体的体外制备及其重组抗体表达载体的构建与瞬时转染表达。方法 以前期获得的OTA杂交瘤细胞株(O16)为研究对象,通过无血清驯化培养的方式开展OTA单克隆抗体的体外制备研究;采用简并引物法获取OTA杂交瘤细胞的单克隆抗体编码基因,在此基础上构建OTA重组全长抗体表达载体以及OTA重、轻链可变区基因片段与人源恒定区片段连接的重组嵌合抗体表达载体,并基于哺乳动物细胞系,开展两种重组抗体的瞬时转染表达研究。结果 通过缓降血清浓度的方式对OTA杂交瘤细胞进行无血清驯化,实现了体外培养制备OTA单克隆抗体;在此基础上测得OTA单克隆抗体的重链和轻链基因序列,并成功构建了OTA重组全长抗体表达载体(pCDNA3.4-OTA-H/L)和重组嵌合抗体表达载体(p FUSE-OTA-H/L),通过共转染实现了两者在哺乳动物细胞系中的瞬时转染表达;经间接酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)确定体外培养制备的OTA抗体、OTA重组全长抗体和嵌合抗体均具备特异性结合抗原的能力,三者的...

Preparation of ochratoxin A monoclonal antibody in vitro and construction of recombinant antibody

Objective To achieve the preparation of anti-ochratoxin A ( OTA ) monoclonal antibody in vitro and the construction of recombinant antibody expression vector and transient transfection expression. Methods The OTA hybridoma cell line (O16) obtained in the previous study was used as the research object, and the preparation of OTA monoclonal antibody in vitro was carried out by serum-free domestication culture. The monoclonal antibody coding gene of OTA hybridoma cells was obtained by degenerate primer method. On this basis, the recombinant full-length antibody expression vector of OTA and the recombinant chimeric antibody expression vector of OTA heavy and light chains connected with human constant region fragments were constructed, and transient transfection expression of the two recombinant antibodies was conducted based on mammalian cell Expi 293F. Results OTA hybridoma cells were acclimated serum-free by slowing down serum concentration, and OTA monoclonal antibodies were prepared in vitro. On this basis, the heavy chain and light chain gene sequences of OTA monoclonal antibody were measured, and the OTA recombinant full-length antibody expression vector (pCDNA3.4-OTA-H/L) and recombinant chimeric antibody expression vector (pFUSE-OTA-H/L) were successfully constructed. The transient transfection expression of the two in mammalian cell lines was realized by co-transfection. The indirect Elisa confirmed that the OTA antibody, OTA recombinant full-length antibody and chimeric antibody prepared in vitro had the ability to specifically bind to the antigen. The linear detection ranges of the three antibodies were 1.043~1.671 ng/mL, 0.9437~1.245 ng/mL, 1.387~1.902 ng/mL, respectively, and all retained the specific binding ability to OTA. Conclusion OTA hybridoma cells adapted to serum-free culture were obtained by slowly decreasing serum concentration, and OTA monoclonal antibodies were prepared by in vitro culture. The expression vectors of OTA full-length recombinant antibody and human chimeric antibody were successfully constructed, and the transient transfection expression of the recombinant antibody was realized in mammalian cells.