动物医学进展2025，Vol.46Issue(8)：32-38,7.
猪白细胞分化抗原32B的原核表达与单克隆抗体制备
Prokaryotic Expression and Monoclonal Antibody Preparation of Porcine CD32B
摘要
Abstract
To prepare monoclonal antibody(mAb)against porcine cluster of differentiation 32B(CD32B),the total RNA of porcine peripheral blood mononuclear cells was used as a template to clone porcine CD32B gene by RT-PCR method.Then the porcine CD32B extracellular region gene fragment without signal pep-tide sequence was inserted into pET-28a(+)vector to construct recombinant expression plasmid,and the porcine CD32B recombinant protein was expressed by Escherichia coli expression system.After being iden-tified by SDS-PAGE and Western blot methods,the targeted protein was expressed in large quantities and purified using Ni-NTA Resin.Balb/c mice were immunized with purified porcine CD32B recombinant pro-tein,the mAb against porcine CD32B was prepared by cell fusion technique,and the obtained mAb was i-dentified.A total length of 894 bp porcine CD32B gene and 537 bp truncated gene were obtained.The con-structed recombinant plasmid pET-28a-pCD32B was transferred into BL21(DE3)competent cells and a 24.6 ku of porcine CD32B recombinant protein was obtained.After multiple rounds of subclonal screening,a hybridoma cell 3E12 line was obtained,which can secrete porcine CD32B mAb stably.The heavy chain and light chain of mAb 3E12 are IgG2α and κ chain,respectively.mAb 3E12 can bind specifically to porcine CD32B recombinant protein expressed by the prokaryotic expression.IFA results showed that mAb 3E12 also can bind to full-length pig CD32B expressed by HEK 293T cells.The acquisition of mAb 3E12 has ac-cumulated material for exploring the role of CD32B in immunosuppressive diseases in pigs.关键词
猪白细胞分化抗原32B/原核表达/蛋白纯化/细胞融合/单克隆抗体Key words
Porcine CD32B/Prokaryotic expression/Protein purification/Cell fusion/Monoclonal antibody分类
农业科技引用本文复制引用
赵治钤,郑丹阳,赵谜,穆杨..猪白细胞分化抗原32B的原核表达与单克隆抗体制备[J].动物医学进展,2025,46(8):32-38,7.基金项目
国家自然科学基金项目(31972675) （31972675）
陕西省动物防疫专项(2023-33) （2023-33）
