Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand.
Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok, Thailand.
Anim Biotechnol. 2022 Jun;33(3):508-518. doi: 10.1080/10495398.2020.1811294. Epub 2020 Aug 31.
Distinguishing between bull Y- and X-bearing sperm populations is advantageous for techniques using sexed bull semen. The aim of this study was to produce a single-chain fragment variable (scFv) antibody against plasma membrane epitopes on bull Y-bearing sperm. Variable heavy (VH)- and variable light (VL)-region genes generated from a hybridoma cell secreting a specific Y-bearing sperm monoclonal antibody (mAb-1F9) were cloned and expressed. The expected sizes of the DNA bands were ∼350 bp for the VH gene and ∼318 bp for the VL gene. The VH and VL genes were generated and used to construct an scFv gene (∼650 bp), which was expressed in TG1 cells and produced the corresponding soluble scFv antibody. Compared with the parent mAb-1F9, the scFv antibodies presented a high affinity for Y-bearing sperm and low cross-reactivity with X-bearing sperm. An immunofluorescence analysis confirmed that the scFv antibodies and mAb-1F9 recognize epitopes on the Y-bearing sperm surface. The fluorescence signal was strong on the plasma membrane of Y-bearing sperm but very weak for X-bearing sperm. This study aids the application and production of engineered scFv antibodies specific to Y-bearing sperm to distinguish between Y- and X-bearing sperm populations for techniques involving sexed bull semen.
区分携带 Y 染色体和 X 染色体的精子群体对于使用性别鉴定公牛精液的技术是有利的。本研究的目的是制备针对携带 Y 染色体精子的质膜表位的单链抗体片段 (scFv)。从分泌特异性携带 Y 染色体精子单克隆抗体 (mAb-1F9) 的杂交瘤细胞中克隆和表达可变重 (VH) 和可变轻 (VL) 区基因。VH 基因和 VL 基因的预期 DNA 带大小分别约为 350 bp 和 318 bp。VH 和 VL 基因被生成并用于构建 scFv 基因（约 650 bp），该基因在 TG1 细胞中表达并产生相应的可溶性 scFv 抗体。与亲本 mAb-1F9 相比，scFv 抗体对携带 Y 染色体的精子具有高亲和力，对携带 X 染色体的精子的交叉反应性低。免疫荧光分析证实 scFv 抗体和 mAb-1F9 识别携带 Y 染色体精子表面的表位。荧光信号在携带 Y 染色体的精子的质膜上很强，但在携带 X 染色体的精子上非常弱。本研究有助于应用和生产针对携带 Y 染色体精子的工程 scFv 抗体，以区分涉及性别鉴定公牛精液的技术中的携带 Y 染色体和 X 染色体的精子群体。
