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

Abstract

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

CLC Number:

S858.285.3

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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References 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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Development of Monoclonal Antibodies against Classical Swine Fever Virus E2 Protein by Single B Cell Sorting Technology and Its Application in ELISA

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