牛呼吸道合胞体病毒G蛋白抗原区原核表达及反应原性鉴定

doi:10.16431/j.cnki.1671-7236.2021.09.036

Abstract

Abstract: The purpose of this study was to obtain recombinant G protein of Bovine respiratory syncytial virus (BRSV) and identify its reactogenicity. The nucleotide sequence of G gene was found from NCBI, its antigenic region was analyzed, and a pair of specific primers was designed with Primer Premier 5.0 software. The BRSV G gene fragment was amplified by RT-PCR and ligated to the cloning vector pMD19-T, and then it was identified by double enzyme digestion and sequenced. The gel was recovered from the target fragment digested by double enzyme, then the recombinant plasmid pET-32a-G was constructed and transformed into E. coli BL21(DE3) competent cells. The protein induced by IPTG was purified by Ni-IDA affinity chromatography and its concentration was determined, then the protein was analyzed and identified by SDS-PAGE and Western blotting. The results showed that the G gene with the size of 567 bp was successfully cloned and the soluble recombinant protein with molecular weight of 40 ku was obtained. After optimizing the induction conditions, the expressed product was identified by SDS-PAGE. The protein expression was the highest at 16 ℃, IPTG concentration 1.2 mmol/L and induction for 4 h. Through Western blotting detection, it found that the recombinant protein could react with goat BRSV standard positive serum specifically, indicating that the protein had reactivity. In summary, BRSV G protein was successfully expressed and purified in this study, which laid a foundation for the establishment of indirect ELISA detection of BRSV antibody and the development of BRSV subunit vaccine.

Key words: Bovine respiratory syncytial virus (BRSV); G protein; prokaryotic expression; soluble expression; reactogenicity

CLC Number:

S852.65⁺3

WU Chunxia, ZHOU Yaping, GUO Ting, CUI Qi, WANG Yuchen, TIAN Guangyuan, Sihan, SUN Yanli, HAO Yongqing. Prokaryotic Expression and Reactogenicity Identification of G Protein Antigen Region of Bovine Respiratory Syncytial Virus[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3438-3446.

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Prokaryotic Expression and Reactogenicity Identification of G Protein Antigen Region of Bovine Respiratory Syncytial Virus

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