猪A3Z2基因生物信息学分析及其对PEDV复制的抑制作用

doi:10.16431/j.cnki.1671-7236.2023.09.026

Abstract

Abstract: 【Objective】 The aim of this study was to perform cloning and bioinformatics of A3Z2 gene of porcine apolipoprotein B mRNA-editing enzyme catalytic polypeptide protein 3B (APOBEC3,A3) family,screen IPEC-J2 cells that stably expressed A3Z2,and investigate the effects of A3Z2 on the replication of Porcine epidemic diarrhea virus(PEDV),in order to lay the foundation for studying the antiviral function of A3Z2.【Method】 Porcine A3Z2 gene was amplified from the total RNA of IPEC-J2 cells as a template,the CDS region of porcine A3Z2 gene was amplified by RT-PCR,and its genetic evolution and bioinformatics analysis were performed.The pcDNA3.1-3×Flag-A3Z2 vector was constructed and transfected into IPEC-J2 cells screened by G418,the stable A3Z2 expressing cell line was obtained using limited dilution method and monoclone cell culture.The expression of A3Z2 was identified using indirect immunofluorescence experiment and Western blotting,the mRNA and protein levels of PEDV N gene were analyzed using Real-time quantitative PCR and Western blotting,respectively.【Result】 The full-length of A3Z2 gene CDS region was 843 bp,encoded 280 amino acids,the molecular weight of A3Z2 protein was 32.7 ku. There was no transmembrane region and signal peptide catting site,mainly in the nucleus. The secondary structure of A3Z2 protein was mainly composed by 27.14% alpha helix,17.86% extended chain,6.43% beta turn and 48.57% random coil.The A3Z2 stable expression cell line IPEC-J2-A3Z2 was obtained through cell screening.The results of indirect immunofluorescence assay and Western blotting showed that A3Z2 expressed in IPEC-J2-A3Z2 cells.The results of Real-time quantitative PCR and Western blotting found that the mRNA and protein expression of PEDV N gene in IPEC-J2-A3Z2 cells were inhibited by A3Z2.【Conclusion】 Porcine A3Z2 gene was successfully cloned and IPEC-J2-A3Z2 cell line was obtained.Moreover,A3Z2 had the antiviral activity of PEDV replication.The results provided insight for studying the molecular mechanism of PEDV and A3Z2,and as a target to screen new anti-PEDV drugs.

Key words: pig; A3Z2 gene; bioinformatics; Porcine epidemic diarrhea virus (PEDV); IPEC-J2 cells

CLC Number:

S852.65⁺1

SHEN Haiyan, WANG Songqi, ZHANG Bin, LIU Zhicheng, ZHANG Jianfeng, LIAO Ming, ZHANG Chunhong. Bioinformatics Analysis of Porcine A3Z2 Gene and Its Antiviral Activity of PEDV Replication[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3695-3706.

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Bioinformatics Analysis of Porcine A3Z2 Gene and Its Antiviral Activity of PEDV Replication

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