猫PD-L1单克隆抗体及猫源化单链抗体的制备和初步应用

doi:10.11843/j.issn.0366-6964.2026.01.035

摘要/Abstract

摘要：

旨在制备猫PD-L1抗原并筛选单抗，探讨其在犬、猫肿瘤检测和诊断中的应用，同时制备猫源化嵌合单链抗体，为猫肿瘤的治疗提供基础。本研究通过PCR扩增猫PD-L1基因，构建原核表达重组质粒PET-MPD-L1，通过在宿主BL21（DE3）中表达制备PD-L1抗原，免疫小鼠后细胞融合，ELISA方法进行杂交瘤细胞筛选，3次亚克隆后建立MPD-L1杂交瘤细胞系。测定杂交瘤细胞上清、腹水效价、抗体亚类、Western blot和免疫组织化学（IHC）特性。扩增轻、重链可变区和猫IgG1 Fc基因，降落PCR将重链可变区（Variable region of the Heavy chain，VH）和轻链可变区（Variable region of the Light chain，VL）拼接成scFv，进一步拼接出scFv-fFc，构建pFast-MPD-L1-scFv和pFast-MPD-L1-scFv-fFc重组质粒，在昆虫杆状病毒表达系统中表达。结果表明，猫PD-L1在上清中大量表达且纯度较好，杂交瘤细胞2A5A2F4D7分泌的抗体属于IgG 2a，轻链为κ亚型，细胞上清、腹水抗体效价分别为1∶3 200和1∶102 400，Western blot结果表明，该单抗能与表达的猫PD-L1反应，IHC结果显示，该单抗可以识别犬猫肿瘤组织细胞表达的PD-L1，可以反映PD-L1在肿瘤组织中的表达情况。VH和VL与IMGT数据库中相应基因序列相符，具有4个框架区和3个高变区，猫源化嵌合单链抗体在昆虫杆状病毒表达系统中成功表达。综上，本研究制备的猫PD-L1单克隆抗体在犬猫肿瘤检测和预后中具有一定的价值，猫源化单链抗体的成功制备为其在犬猫肿瘤病治疗中的应用打下了基础。

关键词: 犬猫肿瘤疾病, 猫PD-L1, 单克隆抗体, scFv, scFv-fFc, 昆虫杆状病毒表达

Abstract:

The purpose of this study was to prepare feline PD-L1 antigen and screen monoclonal antibody， and preliminary application was carried out in detection and diagnosis of canine and feline oncology diseases. In addition， feline derived chimeric single chain variable Fragment-Feline Fragment crystallizable （scFv-fFc） was prepared， which provided a tool for the treatment of cat tumor in future. The PD-L1 gene of feline was amplified by PCR and prokaryotic expression recombinant plasmid PET-MPD-L1 was constructed， and the antigen of PD-L1 was prepared by expression with host BL21（DE3）. After immunization with mice， cell was fused， and an ELISA was established to screen antibody produced by hybridoma cells. After subcloning three times， the cell line of MPD-L1 hybridoma were established， subtypes were identified， and the ascites were prepared， and titer of the ascites was determined. Characteristics were determined by Western blot and immunohistochemistry （IHC）. Total RNA was extracted from the hybridoma cells， and the heavy chain variable region （VH） and light chain variable region （VL） genes were amplified by RT-PCR， which were compared with the antibody light and heavy chain genes in the IMGT database for confirmation. RNA from peripheral blood of a cat in clinic was extracted， and the Fc gene of IgG1 was amplified by RT-PCR. The recombinant plasmids of pFast-MPD-L1-scFv and PFAST-MPD-L1-scFv-fFc were constructed by splicing VH and VL into scFv and scFv-Fc into scFv-fFc by landing PCR， then expressed in the baculovirus expression system. The results showed that feline PD-L1 was highly expressed in the supernatant of cultures and the purified protein could be used antigen for immunization. The monoclonal antibody of 2A5A2F4D7 belonged to IgG 2a subtype， and the light chain is subtype κ. The titer of the supernatant， ascites was 1：3200， 1：102400 respectively. Western-Blot results showed that monoclonal antibody 2A5A2F4D7 reacted with expressed feline PD-L1， and IHC results showed that the monoclonal antibody secreted by 2A5A2F4D7 hybridoma cells can recognize PD-L1 of tumor cells both canine and feline. The VH and VL genes amplified were consistent with genes in the IMGT database， with 4 framework regions and 3 hypervariable regions. The recombinant plasmids of pFast-MPD-L1-scFv and PFAST-MPD-L1-scFv-fFc were constructed successfully and expressed in the supernatants of insect baculovirus expression system. The prepared feline PD-L1 monoclonal antibody have certain value in the detection and prognosis of canine and feline tumors， while scFv-fFc might hold great potential for application in the treatment of tumors in canine and feline.

Key words: tumor diseases in canine and feline, feline PD-L1, monoclonal antibodies, scFv, scFv-fFc, insect baculovirus expression

中图分类号:

S856

李蒙, 苏思元, 李自盼, 徐昀眺, 黄紫贝, 罗健雅, 赵以恒, 郭鑫, 卞泓凯, 潘新语, 刘文博. 猫PD-L1单克隆抗体及猫源化单链抗体的制备和初步应用[J]. 畜牧兽医学报, 2026, 57(1): 401-413.

LI Meng, SU Siyuan, LI Zipan, XU Yuntiao, HUANG Zibei, LUO Jianya, ZHAO Yiheng, GUO Xin, BIAN Hongkai, PAN Xinyu, LIU Wenbo. Preparation and Application of Cat PD-L1 Monoclonal Antibody and Feline Derived Single Chain Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 401-413.

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