牦牛源BVDV非结构蛋白NS5A的原核表达及抗原表位分析

doi:10.16431/j.cnki.1671-7236.2022.08.034

Abstract

Abstract: 【Objective】 The NS5A gene of Bovine viral diarrhea virus (BVDV) was expressed in vitro by prokaryotic expression system to obtain the non-structural protein NS5A.The nucleotide and amino acid sequences were analyzed to analyze the function of BVDV non-structural protein NS5A.【Method】 A pair of primers were designed and synthesized according to the NS5A gene sequence of BVDV-1 V006 strain(GenBank accession No.:KX170647) deposited in GenBank. The NS5A gene was amplified by PCR using the cDNA of BVDV GSTZ strain from yak as a template and was inserted into the pET-28a(+) vector,and the recombinant prokaryotic expression vector pET28a-NS5A was constructed. After preliminary identification by enzyme digestion and sequencing,E.coli BL21 (DE3) competent cells were transformed,and then induced by IPTG.The expression of the recombinant protein was identified by 10% SDS-PAGE electrophoresis and Western blotting analysis,and the genetic development and evolution tree was constructed according to the sequence of NS5A gene.The hydrophilicity,surface plasticity and antigenicity of NS5A protein were predicted by DNAStar software,and the B-cell antigen epitope of NS5A protein was predicted combined with the prediction of secondary structure.【Result】 The target gene fragment of NS5A amplified by PCR was 1 488 bp,which was consistent with the expectation.The results of double enzyme digestion and sequencing showed that the recombinant plasmid pET28a-NS5A was successfully constructed.The recombinant protein was identified by 10% SDS-PAGE electrophoresis and Western blotting,and the target protein with the size of 55 ku was expressed.The size was consistent with the expected results.Through the construction of genetic development evolutionary tree for different BVDV strain NS5A gene sequences,it showed that GSTZ strain NS5A belonged to BVDV-1 in genetic evolutionary characteristics. The hydrophilicity of NS5A protein were mainly located at amino acids 12-21, 32-69, 75-113, 120-135, 143-147, 152-163, 165-180, 215-230, 265-274, 296-340, 348-378, 389-447, 455-463, 469-495. The surface plasticity were mainly located at amino acids 14-18, 37-42, 76-81, 86-109, 154-160, 169-178, 218-228, 297-309, 348-358, 365-373, 414-442, 430-437, 454-460. And there were many flexible regions, mainly located at amino acids 14-21, 37-43, 67-82, 86-93, 97-110, 152-158, 169-179, 218-231, 240-255, 296-310, 313-328, 344-359, 364-373, 413-422 and 472-483. The B-cell antigen epitopes of NS5A protein were mainly located at amino acids 15-18,76-81,154-158,169-178,218-228,297-309,348-358,365-373 and 414-422.【Conclusion】 The non-structural protein NS5A of BVDV from yak was successfully expressed and identified.The phylogenetic tree showed that the BVDV GSTZ isolate was classified into BVDV-1.NS5A protein had good antigenicity.This study provided a basic plateform which provided reference for function and immunological characteristics of BVDV non-structural protein NS5A,and further study on the effect of NS5A protein on viral replication and relationship between biological function and protein structure.

Key words: Bovine viral diarrhea virus; non-structural protein; NS5A; prokaryotic expression

CLC Number:

Q939.94

WANG Huihui, FENG Xili, PU Feiyang, LI Yicong, WANG Mengzhu, ZHOU Xiaokai, ZHAO Zeyang, MA Zhongren, LI Zhuo, MA Xiaoxia. Prokaryotic Expression and Antigen Epitope for Non-structural Protein NS5A of BVDV Isolated from Yak[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3180-3189.

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Prokaryotic Expression and Antigen Epitope for Non-structural Protein NS5A of BVDV Isolated from Yak

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