猪流行性腹泻病毒截短M基因克隆、生物信息学分析及其截短片段表达

doi:10.16431/j.cnki.1671-7236.2022.04.030

Abstract

Abstract: 【Objective】 The aim of this study was to investigate the sequence structure and prokaryotic expression of Porcine epidemic diarrhea virus (PEDV) truncated M protein.【Method】 A pair of specific primers were designed according to the published M gene sequence of PEDV.The truncated M gene (rM) of PEDV was amplified by RT-PCR and cloned using RNA extracted from positive clinical samples.The structural characteristics of rM protein were predicted by online bioinformatics software.The obtained truncated M gene was cloned into prokaryotic expression vector pET-28a(+),and the prokaryotic expression plasmid pET-28a-rM was constructed.The correctly identified pET-28a-rM was transformed into E.coli BL21(DE3) competent cells,and the recombinant strain BL21(pET-28a-rM) was successfully constructed,then the recombinant strain was induced by IPTG,the expression conditions of the recombinant protein were optimized,and the recombinant protein was purified by affinity chromatography and detected by SDS-PAGE and Western blotting.Meanwhile,rabbit anti-rM polyclonal antibody was prepared by using the recombinant protein.【Result】 The truncated M gene with a size of 366 bp was cloned.The recombinant protein was composed of 121 amino acids,and the predicted molecular weight was about 12.8 ku.The secondary structure of the protein was composed of random coil,extended strand,beta turn and alpha helix,and the components were 48.76%,33.88%,9.09% and 8.26%,respectively.The protein contained no signal peptide,but had transmembrane region,containing 21 phosphorylation sites.SDS-PAGE results showed that the size of recombinant protein was about 15 ku and existed with the form of inclusion body protein.The protein expression was the highest when induced at 37 ℃ with 1 mmol/L IPTG for 12 h.Western blotting result showed that the recombinant protein had good reactogenicity with the PEDV positive serum.The high immune serum with the titer higher than 1∶51 200 was obtained after the New Zealand White rabbits immunized with purified recombinant protein.【Conclusion】 This study successfully cloned the PEDV truncated M gene the bioinformatics analysis was carried out,and obtained high-purity rM protein,and established a material foundation for the development of biological products for porcine epidemic diarrhea treatment and detection.

Key words: Porcine epidemic diarrhea virus (PEDV); M gene; bioinformatics analysis; prokaryotic expression

CLC Number:

S852.65^＋1

ZHANG Mingliang, GENG Yachun, LIAN Kaiqi, MA Lei, CHANG Ying, WANG Shuangshan, ZHANG Fuliang. Cloning,Bioinformatics Analysis and Truncated Fragment Expression for Truncated M Gene of Porcine Epidemic Diarrhea Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1479-1487.

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Cloning,Bioinformatics Analysis and Truncated Fragment Expression for Truncated M Gene of Porcine Epidemic Diarrhea Virus

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