Preparation of mouse monoclonal antibodies against the ectodomain of Western equine encephalitis virus E2 (E2ecto) protein.
- Author:
Fuxing WU
1
,
2
;
Yangchao DONG
3
;
Jian ZHANG
3
;
Pan XUE
1
,
2
;
Ruodong YUAN
3
;
Yang CHEN
3
;
Hang YUAN
1
,
2
;
Baoli LI
4
;
Yingfeng LEI
5
Author Information
1. School of Medicine, Yan'an University, Yan'an 716000
2. Department of Microbiology and Pathogenic Biology, Basic Medical Science Academy, Air Force Medical University, Xi'an 710032, China.
3. Department of Microbiology and Pathogenic Biology, Basic Medical Science Academy, Air Force Medical University, Xi'an 710032, China.
4. School of Medicine, Yan'an University, Yan'an 716000, China. *Corresponding authors, E-mail: lbl_0812@163.com.
5. Department of Microbiology and Pathogenic Biology, Basic Medical Science Academy, Air Force Medical University, Xi'an 710032, China. *Corresponding authors, E-mail: yflei@fmmu.edu.cn.
- Publication Type:Journal Article
- MeSH:
Horses;
Animals;
Mice;
Encephalitis Virus, Western Equine;
Ascites;
Immunosuppressive Agents;
Antibodies, Monoclonal;
Immunoglobulin M
- From:
Chinese Journal of Cellular and Molecular Immunology
2024;40(1):62-68
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 105, and 3D5B7 reached 107. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.
