利用单B细胞分选技术制备猪瘟病毒E2蛋白单克隆抗体及其在ELISA中的应用

doi:10.11843/j.issn.0366-6964.2024.10.029

摘要/Abstract

摘要：

本研究旨在建立一种猪瘟病毒E2单抗竞争ELISA检测方法, 用于评价猪瘟C株弱毒苗和E2亚单位疫苗的免疫效果。首先，构建猪瘟E2杆状病毒表达载体pFastBAC，通过悬浮培养SF9细胞高效表达E2蛋白；其次，用纯化的E2蛋白免疫BABL/c小鼠，通过流式分选IgM-PE-/E2-APC+型单个B细胞。然后，利用半巢式PCR分别扩增E2特异性IgG抗体的重链和轻链，测序后将抗体重链及轻链构建到pCDNA3.1载体，再将构建好的质粒共转染至HEK-293细胞, 制备猪瘟E2单克隆抗体。结果表明，本研究通过单B细胞分选技术获得的两株单克隆抗体，即mAb3A9(IgG1, kappa)和mAb4F7(IgG2a, lambda), 分别识别猪瘟E2蛋白B细胞线性表位²⁵GLTTTWKEYSHDLQL³⁹和²⁵⁹GNTTVKVHASDERGP²⁷³。此外，用上述两株单克隆抗体及E2蛋白建立的单抗竞争ELISA检测方法，在血清样本检测过程中，均展现出优异的诊断敏感性(97.49%，95.97%)及特异性(96.08%，94.38%)，该研究为我国猪瘟的逐步净化提供了有利的技术支撑。

关键词: 猪瘟, E2, 单B细胞, 表位, ELISA

Abstract:

The purpose of this study was to develop a classical swine fever virus (CSFV) E2 monoclonal antibody-based competition ELISA (Enzyme Linked Immunosorbent Assay) for evaluating the efficiency of the live attenuated C-strain and E2 subunit vaccines. Firstly, the E2 gene of CSFV was constructed into pFast BAC vector, and the E2 protein was efficiently expressed in SF9 cells; Secondly, 6-8 weeks of BABL/c mice were immunized at intervals with purified E2 protein, and the single B cells were then sorted out at the gate of IgM-/E2+ by flow cytometry. Then, the heavy and light chains of E2 antigen-specific IgG antibodies were amplified by semi-nested PCR, after sequencing, the heavy and light chains of the antibodies were constructed into pCDNA3.1vector, and then were co-transfected into HEK293 cells to prepare the monoclonal antibodies against CSFV E2. The results showed that the two monoclonal antibodies derived from single B cells, namely mAb3A9 (IgG1, kappa) and mAb4F7 (IgG2a, lambda), could efficiently reacted with the linear epitopes²⁵GLTTTWKEYSHDLQL³⁹ and²⁵⁹GNTTVKVHASDERGP²⁷³ of CSFV E2 protein, respectively. In addition, the competitive ELISAs developed using the monoclonal antibodies and E2 protein mentioned above exhibited an excellent diagnostic sensitivity (97.49%, 95.97%) and specificity (96.08%, 94.38%) in the process of detecting serum samples, which provides a favorable technical support for the gradual elimination of CSF in China.

Key words: classic swine fever, E2, single B cells, epitopes, ELISA

中图分类号:

S858.285.3

马中元, 郑君佐, 梁志博, 潘丽, 曾巧英. 利用单B细胞分选技术制备猪瘟病毒E2蛋白单克隆抗体及其在ELISA中的应用[J]. 畜牧兽医学报, 2024, 55(10): 4579-4589.

Zhongyuan MA, Junzuo ZHENG, Zhibo LIANG, Li PAN, Qiaoying ZENG. Development of Monoclonal Antibodies against Classical Swine Fever Virus E2 Protein by Single B Cell Sorting Technology and Its Application in ELISA[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4579-4589.

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参考文献 21

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引物名称 Primer name	引物序列(5′→ 3′) Sequence
Igγ forward	AGAGTGCTGATTCTTTTGTG
	GAATGGCCTTATATCTTTCTC
	GAATGGAGTTGGATATTTCTC
	TCCTTGAATCTTTCTCTTCCT
	GGATTCAGCAGGATCTTTCTC
	GGGACACAGGACCTCACCAT
	GGATGGAGCTGTATCATCCTC
	GGATGGAGCTGTTTCATCGTC
	GGATTGCTGTTTCTTTTCTTC
	GAATGGCTGTGGAACTTGCT
Igγ reverse outer	GAAGGTGYGCACACYGCTGGAC (1^st PCR)
Igγ reverse inner	GCTCAGGGAARTAGCCCTTGAC (2^nd PCR)

引物名称 Primer name	引物序列(5′→ 3′) Sequence
Igλ forward	GAGACAGACACAATCCTGCTA
	GATTTTCAAGTGCAGATTTTC
	AAGTTGCCTGTTAGGCTGTTG
	GATTCACAGGCCCAGGTTCTT
	GAATCACAGACTCAGGTCCTC
	TTACTGCTGCTATGGGTATCT
	ACCATGTTCTCACTAGCTCTT
	TTCTGGATTCCTGCTTCCAGC
	AGGGTCCTTGCTGAGCTCCTG
	GAGACAGACACACTCCTGTTA
	GAGACAGACACACTCCTGCTA
	ACCATGCTCTCACTAGCTCCT
Igλ reverse outer	GTACCATYTGCCTTCCAGKCCACT (1^st PCR)
Igλ reverse inner	CTCYTCAGRGGAAGGTGGRAACA (2^nd PCR)

引物名称 Primer name	引物序列(5′→3′) Sequence
Igκ forward	GCTGTTTTGTATACCTGGG
	CTCAGCTCCTGTTGCTGTGGC GTGTMTGGTGCTBRT
	GGGTATCTGGTACCTGTGG
	CCTCATATTTTTGCTGCTATGGG
	TGCTTTTCTGGATTTCAGCCTCC
	TCAACTTCTGCTCTTCCTGCTGT
	CTAGCTCYTCTCCTCAGYCTTC
Igκ reverse outer	ACTGAGGCACCTCCAGATG (1^st PCR)
Igκ reverse inner	TGGGAAGATGGATACAGTT (2^nd PCR)

方法 Methods	结果 Results	mAb3A9-bELISA			mAb4F7-bELISA
方法 Methods	结果 Results	阳性 Positive No.	阴性 Negative No.	合计 Total	阳性 Positive No.	阴性 Negative No.	合计 Total
IDEXX	阳性Positive	56	4	60	54	6	60
	阴性Negative	2	34	36	3	33	36
合计Total		58	38		57	39
符合率Coincidence rate		56/60	34/36	93.75%	54/60	33/36	90.62%

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