Original Article
The generation and characterization of the polyclonal antibody against Coxsackievirus B3 3C protease
Niu Sunmin, Zhang Jian, Wang Lixin, Wang Yao, Zhao Shuoxuan, Wo Xiaoman, Zhao Wenran
Published 2023-05-05
Cite as Int J Immunol, 2023, 46(3): 233-239. DOI: 10.3760/cma.j.issn.1673-4394.2023.03.001
Abstract
ObjectiveTo generate and characterize the polyclonal antibody against the 3C protease of Coxsackievirus B3 (CVB3).
MethodsThe DNA of 3C was amplified from pcDNA3.1(+ )-EGFP-3C by polymerase chain reaction. The pET28a(+ )-3C-6His plasmid, which expresses His-tagged 3C, was constructed and used to transform Escherichia coli(E.Coli) BL21(DE3). The expression conditions and the extraction protocol for the recombinant 3C(3C-6His) protein were optimized. After ultrasonic treatment, the recombinant 3C expressed in the bacteria was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The gel was stained by potassium chloride and sliced to collect the proteins with the relative molecular weight of 26 000, which corresponds to the recombinant 3C-6His (26 000). New Zealand white rabbits were immunized with 1 mg of the purified 3C recombinant protein in combination with equal amount of Freunds adjuvant for a total of 5 times, with an interval of one week. One week after the final immunization, the sera of the rabbits were collected to test the characterization of the antibody.
ResultsThe expression of 3C-6His fusion protein was detected at a relative molecular weight of 26 000.The plasmid expressing the His-tagged recombinant 3C protein was constructed, and the recombinant 3C was expressed with high quality.The optimal expression condition was established, in which E. Coli were cultured at 37 ℃ for 6 h with the addition of 0.05 mmol/L sopropyl β- D-thiogalactoside(IPTG).High quantity of recombinant 3C protein was obtained after SDS-PAGE separation.The obtained rabbit antisera can bind 3C protein expressed in E. Coli and HeLa cells.The anti-3C antiserum can also recognize the 3C protease expressed in HeLa cells infected with CVB3 or enterovirus A71.
ConclusionA polyclonal antibody against CVB3 3C protease was obtained, which can specifically bind to the 3C protease of enterovirus. This result maybe provided the basis for the further study about the role played by 3C protease in the pathogenesis of enteroviruses.
Key words:
Coxsackievirus B; 3C protease; Polyclonal antibody
Contributor Information
Niu Sunmin
Department of Cell Biology, Harbin Medical University, Harbin 150081, China
Zhang Jian
Department of Cell Biology, Harbin Medical University, Harbin 150081, China
Wang Lixin
Department of Cell Biology, Harbin Medical University, Harbin 150081, China
Wang Yao
Department of Cell Biology, Harbin Medical University, Harbin 150081, China
Zhao Shuoxuan
Department of Cell Biology, Harbin Medical University, Harbin 150081, China
Wo Xiaoman
Department of Cell Biology, Harbin Medical University, Harbin 150081, China
Zhao Wenran
Department of Cell Biology, Harbin Medical University, Harbin 150081, China
