新型阿卡斑病毒NSs蛋白生物信息学分析及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.05.023

Abstract

Abstract: 【Objective】 This study was aimed to perform the bioinformatics analysis of non-structura (NSs) protein of novel Akabane virus (AKAV) and prepare anti-polyclonal antibody,so as to provide necessary materials for detection and functional study of novel AKAV.【Method】 The novel AKAV NSs protein was systematically analyzed using some online analysis website for protein bioinformatics.The full-length NSs gene was cloned and the recombinant NSs protein was expressed by the expression system of Escherichia coli.The induction conditions of the recombinant protein were optimized and the solubility of the recombinant protein was analyzed.The crushed Escherichia coli expressing NSs protein was collected,denatured by 8 mol/L urea,and purified by Ni²⁺-NTA affinity chromatography.After dialysis and concentration,the purified NSs protein was identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared by immunizing New Zealand White rabbits with purified recombinant NSs protein,then the titer and specific reactivity of the polyclonal antibody were determined by indirect ELISA,Western blotting and indirect immunofluorescence assay (IFA).【Result】 Bioinformatics analysis results showed that the NSs protein was composed of 91 amino acid residues,with an isoelectric point of 12.10,belonging to basic protein.Its instability index was about 79.04,and the average hydrophilicity index was ―0.059,indicating that it was an unstable hydrophilic protein.There was no transmembrane region and signal peptide,the NSs protein was a non-secreted protein.The NSs protein prokaryotic expression vector pET-32a-NSs was successfully constructed.The results of SDS-PAGE and Western blotting showed that the recombinant NSs protein was successfully expressed,with a molecular weight of 29.3 ku and mainly existed in the form of inclusion body,which was consistent with the results of bioinformatic analysis.The titer of the prepared polyclonal antibody reached 1∶16 384 000.The results of Western blotting and IFA showed that the prepared antibody could react specifically with NSs protein,having good specificity.【Conclusion】 The novel AKAV NSs protein was successfully purified and showed good immunogenicity.The polyclonal antibody with high titer and good specificity was prepared,which laid a foundation for the development of vaccine,disease detection and the function study of NSs protein.

Key words: novel Akabane virus (AKAV); NSs protein; bioinformatics; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺9.5

CAI Chang, CHEN Hewei, SONG Ruipeng, QIN Shaomin, LIU Jinfeng, QIN Shuying, SUN Qian, CHEN Tongjinyue, OU Changbo, LIU Xingyou, MA Ling, WU Jianmin. Bioinformatics Analysis and Preparation of Polyclonal Antibody of NSs Protein of Novel Akabane Virus[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 2015-2026.

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Bioinformatics Analysis and Preparation of Polyclonal Antibody of NSs Protein of Novel Akabane Virus

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