表达猪繁殖与呼吸综合征病毒N蛋白的重组腺病毒的构建及其免疫原性分析

doi:10.16431/j.cnki.1671-7236.2022.03.024

Abstract

Abstract: 【Objective】 The aim of this study was to express the nucleocapsid protein of Porcine reproductive and respiratory syndrome virus (PRRSV) by Adenovirus AdMax system expression vector, and to study its immunogenicity.【Method】 Referring to the PRRSV N gene sequence published in GenBank (accession No.:KT945017.1), the PRRSV N gene was synthesized and connected to the Adenovirus shuttle vector pDC316-mCMV-EGFP, transformed into E.coli Top10 competent cells, and the recombinant shuttle plasmid pDC316-N was constructed.The recombinant shuttle plasmid pDC316-N and the framework plasmid PBHGLOX(delta)E1, 3Cre of AdMax Adenovirus system were co-transfected into 293A cells to obtain the recombinant Adenovirus rAd-N.The obtained recombinant Adenovirus solution was identified by PCR and sequencing.The 293A cells were infected with the identified correct rAd-N virus solution.The recombinant Adenovirus was expanded for culture and the TCID₅₀of the virus was detected, RT-PCR and Western blotting were used to detect the expression and reactogenicity of recombinant Adenovirus.Mice were immunized with recombinant Adenovirus, the serum was collected, the antibody level was detected with PRRSV antibody detection kit, and the immune effect on mice was preliminarily evaluated.【Result】 A 400 bp PRRSV N gene band was amplified by PCR.The sequencing results were correct, indicating that the recombinant Adenovirus was successfully constructed.After concentration, it was determined that its TCID₅₀ was 10^-10.239.The results of RT-PCR and Western blotting confirmed that the target gene was correctly expressed at the gene and protein levels, and the molecular weight of the protein was about 14 ku.The detection of mouse specific antibody showed that mice immunized with recombinant Adenovirus rAd-N could quickly produce specific antibody against PRRSV, which was significantly different from the control group (P<0.05).The effect of recombinant Adenovirus combined with Gel adjuvant was the best, which was up to 7.84 U/L.【Conclusion】 The recombinant Adenovirus expressing PRRSV N protein was successfully constructed, which had good immunogenicity, and laid a foundation for the establishment of indirect ELISA for PRRSV antibody and the further development of PRRSV antibody detection kit.

Key words: Porcine reproductive and respiratory syndrome virus (PRRSV); nucleocapsid protein; recombinant Adenovirus; immunogenicity

CLC Number:

S852.65⁺1

LIU Qingqing, WU Peng, LI Peidong, ZHANG Jiangwei, CHEN Chuangfu, XIAO Chencheng. Construction of Recombinant Adenovirus Expressing Porcine Reproductive and Respiratory Syndrome Virus N Protein and Its Immunogenicity Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1024-1031.

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Construction of Recombinant Adenovirus Expressing Porcine Reproductive and Respiratory Syndrome Virus N Protein and Its Immunogenicity Analysis

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