Abstract

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 E.coli 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.

摘要

区分公牛 Y 和 X 精子群对于使用有性公牛精液的技术是有利的。本研究的目的是产生一种针对公牛 Y 精子上的质膜表位的单链片段可变 (scFv) 抗体。克隆和表达由分泌特定 Y 携带精子单克隆抗体 (mAb-1F9) 的杂交瘤细胞产生的可变重 (VH) 和可变轻 (VL) 区基因。VH 基因的 DNA 条带的预期大小约为 350 bp，VL 基因约为 318 bp。产生 VH 和 VL 基因并用于构建在大肠杆菌中表达的 scFv 基因（~650 bp）TG1 细胞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 的精子具有高亲和力，而与携带 X 的精子的交叉反应性较低。免疫荧光分析证实 scFv 抗体和 mAb-1F9 识别带有 Y 的精子表面上的表位。携带Y精子的质膜上的荧光信号强，而携带X精子的荧光信号非常弱。本研究有助于应用和生产对携带 Y 的精子具有特异性的工程化 scFv 抗体，以区分携带 Y 和 X 的精子群体，以用于涉及有性公牛精液的技术。