新型鸭呼肠孤病毒DH13株结构蛋白σB的原核表达及免疫原性分析

doi:10.16431/j.cnki.1671-7236.2021.11.030

Abstract

Abstract: The purpose of this study was to express recombinant σB protein of DH13 strain of Novel duck reovirus (NDRV) using prokaryotic expression system, and detect its immunogenicity, so as to provide material basis for the development of NDRV gene engineering vaccine. σB gene of NDRV DH13 strain was amplified by RT-PCR and prokaryotic expression recombinant plasmids pET-30a-σB and pET-32a-σB were constructed and transformed into E. coli BL21 (DE3) and with IPTG induction corresponding recombinant proteins were obtained and analyzed by SDS-PAGE. The recombinant protein without His tag was purified by digestion and as immunogen to immunize New Zealand White rabbits to prepare polyclonal antibody. The recombinant protein with His tag was purified by Ni²⁺ affinity chromatography and as detection antigen to measure rabbit polyclonal antibody titer by indirect ELISA. It was determined whether the polyclonal antibody specifically recognize the recombinant protein by Western blotting. The results showed that σB gene with a size of about 1 100 bp was amplified. The results of SDS-PAGE showed that two recombinant σB proteins were efficiently expressed, the sizes of which were about 41 and 46 ku respectively, and both existed in the form of inclusion bodies. The indirect ELISA results showed the titer of the prepared polyclonal antibody was up to 1:204 800, it could specifically recognize the recombinant protein, which further reflected that the recombinant His σB protein had good immunogenicity. Western blotting results showed that the recombinant σB protein could reacted with the rabbit polyclonal antibody, indicating that the recombinant σB protein expressed by prokaryotic expression had good reactivity. This experiment successfully expressed the recombinant σB protein using the prokaryotic expression system, and confirmed that the NDRV σB protein had good immunogenicity and reactogenicity. This result would help the subsequent identification of the biological function of the NDRV σB protein, preparation of diagnostic antigens and development of new NDRV vaccines.

Key words: Novel duck reovirus (NDRV); σB protein; prokaryotic expression; immunogenicity analysis

CLC Number:

S852.65⁺7

YANG Huihu, HUANG Huilan, YUAN Sheng, LI Wenjun, YAO Xinyan, ZHANG Yuqian, LIANG Zhaoping, HUANG Shujian, ZHANG Xuelian. Prokaryotic Expression and Immunogenicity Analysis of the Structural Protein σB of a Novel Duck Reovirus DH13 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4204-4212.

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Prokaryotic Expression and Immunogenicity Analysis of the Structural Protein σB of a Novel Duck Reovirus DH13 Strain

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