摘要
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,三者的线性检测范围分别为1.043~1.671、0.944~1.245、1.387~1.902 ng/mL,均保留了对OTA的特异性结合能力。结论通过缓降血清的方式得到了适应无血清培养的OTA杂交瘤细胞,进而实现了体外培养制备OTA单克隆抗体;成功构建了OTA全长重组抗体及人源嵌合抗体的表达载体,并在哺乳动物细胞中实现了其重组抗体的瞬时转染表达。
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.Based on it,the recombinant full-length antibody expression vector of OTA and the recombinant chimeric antibody expression vector were constructed,in which the mouse heavy and light chain variable regions were connected with the human constant region fragment.Transient transfection expression of the two recombinant antibodies was conducted based on mammalian cell.Results OTA hybridoma cells were acclimated serum-free by slowing down serum concentration,and OTA monoclonal antibodies were prepared in vitro.Because of this,the heavy chain and light chain gene sequences of OTA monoclonal antibody were measured.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 both in mammalian cell lines was achieved by co-transfection.The indirect enzyme linked immunosorbent assay(ELISA)confirmed that the OTA antibody prepared in vitro,OTA recombinant full-length antibody and chimeric antibody had the ability to specifically bind to the antigen.Their linear detection ranges were 1.043-1.671,0.944-1.245,1.387-1.902 ng/mL,respectively.They all retained the specific binding ability to OTA.Conclusion OTA hybridoma cells adapted to serum-free culture are obtained by slowly decreasing serum concentration,and OTA monoclonal antibodies are prepared by vitro culture.The expression vectors of OTA full-length recombinant antibody and human chimeric antibody are successfully constructed,and
作者
李蓉芳
吴箫
王何瑶
盛玲
张珍
何庆华
LI Rong-Fang;WU Xiao;WANG He-Yao;SHENG Ling;ZHANG Zhen;HE Qing-Hua(State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330000,China;College of Food Science&Technology,Nanchang University,Nanchang 330000,China)
出处
《食品安全质量检测学报》
CAS
北大核心
2023年第6期118-126,共9页
Journal of Food Safety and Quality
基金
国家重点研发计划项目(2018YFC1602203)
江西省主要学科学术和技术人带头人培养计划项目(20194BCJ22003)。
关键词
赭曲霉毒素A
单克隆抗体
重组抗体
真核表达
ochratoxin A
monoclonal antibody
recombinant antibody
eukaryotic expression
