牛嵴病毒单抗制备及双抗体夹心ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2025.10.028

摘要/Abstract

摘要：

本研究旨在制备牛嵴病毒(bovine kobuvirus, BKV)单克隆抗体，并建立一种双抗体夹心ELISA检测方法。利用pET-30a(+)载体，原核表达BKV结构蛋白VP0，VP3和VP1，纯化后分别免疫BALB/c小鼠，三次免疫后分离小鼠脾细胞，与SP2/0细胞融合制备杂交瘤细胞，通过间接ELISA方法筛选，以制备的单克隆抗体为捕获抗体和检测抗体，优化抗体浓度和孵育时间等条件，建立特异性检测BKV的双抗体夹心ELISA方法。结果显示：获得2株稳定分泌单克隆抗体的杂交瘤细胞，分别命名为2A12和5E11，均为抗VP1蛋白抗体；叠加ELISA及抗体可变区序列分析结果表明，其识别不同的抗原位点；单抗特异性检测结果显示，2株单抗特异性结合BKV，不与其它牛腹泻病毒结合；分别用RT-PCR方法和本研究建立的双抗体夹心ELISA方法，对107份临床样品进行检测，结果显示双抗体夹心ELISA方法与RT-PCR方法的阳性符合率为92.2%，阴性符合率为95.3%，总符合率为93.5%。综上，本研究制备了2株BKV单克隆抗体，并建立BKV的双抗体夹心ELISA方法，该方法特异性、重复性和敏感性良好，为BKV的快速诊断与防控奠定了基础。

关键词: 牛嵴病毒, 结构蛋白, 单克隆抗体, 双抗体夹心ELISA

Abstract:

This study aimed to prepare monoclonal antibody against bovine kobuvirus (BKV) and establish a double antibody sandwich ELISA method for detection. The pET-30a (+) vector was utilized to express the BKV structural proteins VP0, VP3, and VP1 in a prokaryotic expression system. Three structural proteins were purified and used for immunization in BALB/c mice respectively. After three immunizations, the spleen cells of the mice were isolated and fused with SP2/0 cells to prepare hybridoma cells. Then the hybridoma cells were screened by the indirect ELISA method, the prepared monoclonal antibodies were used as capture antibody and detection antibody respectively, the conditions such as antibody concentration and incubation time were optimized, and a double antibody sandwich ELISA method for specifically detecting BKV was established. Results were as follows: Two hybridoma cell lines stably secreting monoclonal antibodies were obtained, designated 2A12 and 5E11, both of which were anti-VP1 protein antibodies; The results of the superimposed ELISA and antibody variable region sequence analysis indicated that they recognized different antigen sites. The results of monoclonal antibody specificity detection showed that the two monoclonal antibodies were specifically bound to BKV and were not bound to other bovine diarrhea viruses. One hundred and seven clinical samples were detected by RT-PCR method and the double antibody sandwich ELISA method established in this study. The results demonstrated that the positive coincidence rate between the two methods was 92.2%, the negative coincidence rate was 95.3%, and the overall coincidence rate was 93.5%. In conclusion, two monoclonal antibodies against BKV were prepared in this study, and a double antibody sandwich ELISA method for BKV was established. This method has good specificity, repeatability and sensitivity, which lays a foundation for the rapid diagnosis, prevention and control of BKV.

Key words: bovine kobuvirus, structural protein, monoclonal antibody, double antibody sandwich ELISA

中图分类号:

S852.659.6

曹恒志, 马芊玥, 姜艳平, 崔文, 李佳璇, 乔薪瑗. 牛嵴病毒单抗制备及双抗体夹心ELISA方法的建立[J]. 畜牧兽医学报, 2025, 56(10): 5084-5094.

CAO Hengzhi, MA Qianyue, JIANG Yanping, CUI Wen, LI Jiaxuan, QIAO Xinyuan. Preparation of Monoclonal Antibody against Bovine Kobuvirus and Establishment of Double Antibody Sandwich ELISA Method[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5084-5094.

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增片段大小/bp Length of amplified fragment	退火温度/℃ Annealing temperature
BKV-VP0-F	CGGGATCCATGGGCACGAGCCAGACC	1 104	56
BKV-VP0-R	CGAGCTCTCACTGCTTGGTCACATGCC
BKV-VP3-F	CGGGATCCATGCACTGGAAGACTCGAGCC	669	56
BKV-VP3-R	CGAGCTCTCATTGGAGAGCTAGCGTCGG
BKV-VP1-F	CGGGATCCATGGCTGGTGAGGGCATCG	801	54
BKV-VP1-R	CGAGCTCTCATTGGCGCCGTACCAT

引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增片段大小/bp Length of amplified fragment
VH-F	GGCCTCGAGATGCAGGTGCAGTTGATGGAGTCAG	340
VH-R	GTGAATTCTGAGGAGACGGTGACTGAGGT
VL-F	AAGCACGCGTAGACATTGTGCTGACCCAATCTC	340
VL-R	CCAGCGGCCGCGGATACAGTTGGTGCAGCATC

单克隆抗体 monoclonal antibody	说明 Description	相似性/% Per.identity	登录号 Accession No.
2A12 VL	Mus musculusstrain BALB/c isolate 6A truncated immunoglobulin light chain variable region (IgKappa) mRNA, partial cds	99.39	FJ233898.1
5E11 VL	Mus musculusisolate ms6_0280_IGKV3-4_IGKJ1_IGKC immunoglobulin light chain variable region mRNA, partial cds	97.41	PP322127.1
2A12 VH	Mus musculusclone Flu002_d8-1_GC_IgH-305 immunoglobulin heavy chain variable region (Igh) mRNA, partial cds	95.85	KU257208.1
5E11 VH	Mus musculus clone 8G2.6 anti-phosphocholine immunoglobulin heavy chain variable region gene, partial cds	99.28	AY194185.1

组别Group	HRP-2A12
2A12	0.239±0.032
5E11	0.509±0.013
PBS	0.516±0.015

样本 Samples	板内变异系数 Intra-plate variation coefficient		批间变异系数 Inter batch variation coefficient
样本 Samples	${\bar x}$ ± s	CV/%	${\bar x}$ ± s	CV/%
阳性样本 Positive samples	0.763 1±0.027 2	3.57	0.748 5±0.045 8	6.12
	0.739 2±0.060 7	8.21	0.745 3±0.054 6	7.32
	0.741 7±0.037 7	5.08	0.753 7±0.048 3	6.41
	0.748 0±0.041 9	5.60	0.749 2±0.049 6	6.62
阴性样本 Negative samples	0.135 0±0.007 2	5.35	0.128 9±0.010 6	8.24
	0.123 5±0.010 1	8.20	0.136 1±0.008 7	6.36
	0.121 9±0.004 7	3.89	0.124 1±0.010 1	8.12
	0.126 8±0.007 3	5.76	0.129 7±0.009 8	7.56