小鼠细小病毒NS1蛋白的原核表达、多克隆抗体制备及其免疫原性评价

doi:10.16431/j.cnki.1671-7236.2024.11.027

Abstract

Abstract: 【Objective】 The objective of this experiment was to express nonstructural protein 1 (NS1) of Minute virus of mice (MVM) through prokaryotic expression system and prepare polyclonal antibody，which provided research materials for the study of biological function of MVM NS1 protein and the establishment of related detection methods. 【Method】 MVM NS1 gene was linked to plasmid pET-N-His-C-His through homologous recombination to construct recombinant plasmid,which was transformed into Escherichia coli BL21 (DE3) competent cells for induced expression.Soluble proteins without renaturation were screened and purified by different induction conditions.The purified recombinant protein NS1 was immunized with New Zealand White rabbits and polyclonal antibodies were prepared.The titer of the antibodies was determined by indirect ELISA.Western blotting and indirect immunofluorescence assay (IFA) were used to determine the expression and immunogenicity of the recombinant protein. 【Result】 SDS-PAGE analysis showed that the recombinant protein was 76 ku in size,and the optimal expression was achieved under the conditions of IPTG concentration of 0.6 mmol/L and induction temperature of 28 ℃.Indirect ELISA results showed that the titer of the prepared polyclonal antibody was 1∶32 000.Western blotting and IFA detection results showed that the prepared NS1 polyclonal antibody could bind specifically to NS1 protein and MVM. 【Conclusion】 This study successfully expressed and purified MVM NS1 protein,and successfully prepared polyclonal antibody with good immunogenicity,which laid a foundation for further study of the biological function of NS1 protein and its role in the mechanism of MVM replication infection,and the establishment of clinical diagnostic methods for MVM.

Key words: Minute virus of mice; nonstructural protein 1; polyclonal antibody; immunogenicity

CLC Number:

S852.65⁺9.2

ZHANG Xuliang, CHEN Li, ZHAO Zhigang, MA Chang, YOU Jinwei, DONG Min, YUN Shifeng. Prokaryotic Expression,Preparations of Polyclonal Antibodies and Immunogenicity Evaluation of NS1 Protein of Minute Virus of Mice[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(11): 4923-4931.

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Prokaryotic Expression,Preparations of Polyclonal Antibodies and Immunogenicity Evaluation of NS1 Protein of Minute Virus of Mice

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