Thongkham Marninphan, Hongsibsong Surat, Mekchay Supamit, Sathanawongs Anucha, Cao Xue-Ming, Xu Zhen-Lin, Sringarm Korawan
Livestock Innovative and Biocirculation Laboratory, Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai, 50200, Thailand.
School of Health Sciences Research, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand.
Sci Rep. 2025 May 2;15(1):15445. doi: 10.1038/s41598-025-00587-1.
The aim of this study was to construct and produce a single-chain fragment variable (scFv) antibody targeting the plasma membrane epitope on porcine Y-chromosome-bearing sperm (Y-sperm). The hybridoma cloned from 4D1-E8 exhibited the highest specificity, and Y-sperm was used to generate the scFv gene. The expected combination of 450 bp (VH) and 300 bp (VL) resulted in a 795 bp scFv gene. The scFv gene was inserted into the pET22b expression vector and expressed in E. coli BL21 (DE3). The resulting H4:L4 clone produced a highly specific scFv antibody to Y-sperm. The reactivity of the H4:L4 scFv antibody to porcine Y-sperm was confirmed via ELISA and flow cytometry. The H4:L4 scFv antibody exhibited low cross-reactivity with X-sperm (4.14%). The soluble H4:L4 scFv antibody exhibited significantly less cross-reactivity with X-sperm than did the 4D1-E8 mAb (4.14% vs. 15.5%). However, H4:L4 scFv and the 4D1-E8 mAbs had high cross-reactivity with other conventional livestock semen. The scFv antibodies and mAbs were detected on the Y-sperm surface via immunofluorescence, and the fluorescence intensities were particularly strong on the plasma membranes of Y-sperm. In this study, the production of a scFv antibody against porcine Y-sperm was successful and represents a novel achievement. This scFv antibody had a high affinity for porcine Y-sperm. The soluble scFv antibody and mAb can be used to sort Y- and X-sperm in treatments involving porcine semen bearing X or Y chromosomes.
本研究的目的是构建并生产一种靶向猪Y染色体精子（Y精子）质膜表位的单链可变片段（scFv）抗体。从4D1-E8克隆的杂交瘤表现出最高的特异性，并用Y精子产生scFv基因。预期450 bp（VH）和300 bp（VL）的组合产生一个795 bp的scFv基因。将scFv基因插入pET22b表达载体并在大肠杆菌BL21（DE3）中表达。所得的H4:L4克隆产生了一种对Y精子具有高度特异性的scFv抗体。通过酶联免疫吸附测定（ELISA）和流式细胞术证实了H4:L4 scFv抗体对猪Y精子的反应性。H4:L4 scFv抗体与X精子的交叉反应性较低（4.14%）。可溶性H4:L4 scFv抗体与X精子的交叉反应性明显低于4D1-E8单克隆抗体（4.14%对15.5%）。然而，H4:L4 scFv和4D1-E8单克隆抗体与其他传统家畜精液具有高交叉反应性。通过免疫荧光在Y精子表面检测到scFv抗体和单克隆抗体，并且在Y精子的质膜上荧光强度特别强。在本研究中，成功生产了一种针对猪Y精子的scFv抗体，这代表了一项新成就。这种scFv抗体对猪Y精子具有高亲和力。可溶性scFv抗体和单克隆抗体可用于在涉及携带X或Y染色体的猪精液的处理中对Y精子和X精子进行分选。
