猪瘟病毒E2-GM-CSF融合蛋白在HEK293T细胞中的表达和免疫原性分析

doi:10.16431/j.cnki.1671-7236.2022.02.029

Abstract

Abstract: 【Objective】 A HEK293T cell pool expressing E2-GM-CSF fusion protein was established to evaluate the immunogenicity of E2-GM-CSF fusion protein, in order to provide a new option for the development of Classical swine fever virus (CSFV) subunit vaccine.【Method】 Firstly, E2-GM-CSF fusion gene was amplified by PCR and cloned into the pCDH lentivector.Then, the recombinant plasmid pCDH-E2-GM-CSF was constructed and packaged into lentiviral particles.Secondly, recombinant HEK293T cell pools expressing E2-GM-CSF fusion protein were obtained by the transduction of the lentiviral particles and the selection of puromycin.The immunogenicity of the purified E2-GM-CSF fusion protein was evaluated in mice.【Result】 The pCDH-E2-GM-CSF plasmid was identified by double-enzyme digestion, and the vector fragments and E2-GM-CSF gene fragments with sizes of 8 172 and 1 521 bp were obtained respectively, indicating that E2-GM-CSF gene was successfully cloned into pCDH vector.A 1 686 bp band was obtained by PCR amplification of cells genomic DNA, which showed that E2-GM-CSF gene was integrated into the genomic DNA of HEK293T cells.Western blotting analysis obtained a band of about 70 ku, proving the successful expression of E2-GM-CSF fusion protein.The SDS-PAGE verification showed that the purified E2-GM-CSF fusion protein was a single band about 70 ku with high purity.The immunization trail showed the good immunogenicity of E2-GM-CSF fusion protein produced by HEK293T cells.The E2 specific antibody of 1∶300 was detected in mouse serum after 14 days of immunization, and the titer of antibody reached 1∶900 after 21 days of immunization.At the 28th day post immunization, the E2 specific antibody titer reached a maximum of 1∶8 100, which was much higher than 1∶1 800 of E2 protein.【Conclusion】 In this study, a HEK293T cell pool expressing E2-GM-CSF fusion protein was established, and the immunogenicity of E2-GM-CSF fusion protein was preliminarily confirmed, providing a novel candidate subunit vaccine against CSFV.Therefore, the development of this vaccine could also provide a reference for the rapid expression of other subunit vaccines in mammalian cells.

Key words: Classical swine fever virus (CSFV); E2-GM-CSF fusion protein; lentiviral vector expression system; HEK293T cells; immunogenicity

CLC Number:

S852.65⁺1

ZHANG Yanmin, ZHOU Yanan, CAO Lei, TIAN Yuan, LIU Xuping, TAN Wensong, ZHAO Liang. Expression and Immunogenicity Analysis of Classical Swine Fever Virus E2-GM-CSF Fusion Protein in HEK293T Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 669-676.

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Expression and Immunogenicity Analysis of Classical Swine Fever Virus E2-GM-CSF Fusion Protein in HEK293T Cells

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