关键词: 猪嵴病毒; 3C基因; 原核表达; 蛋白结构预测

Abstract: In order to obtain the 3C protein of porcine kobuvirus and study its crystal structure, 3C gene was amplified using RT-PCR method with the RNA as template extracted from swKoV CH441 strain, and then was inserted into the pMD19-T Simple Vector.The plasmids were sequenced after verification by PCR and double enzyme digestion.The target fragment was cleaved from the correct plasmid, and was inserted into the pET-30a vector.The recombinant plasmid was transformed into E.coli BL21 (DE3) cell, and its expression was detected after IPTG induction.According to the sequencing results, the full length of the 3C gene was 576 bp, which encoded 192 amino acids.The results of SDS-PAGE suggested that recombinant protein was expressed at the highest level after 0.5 mmol/L IPTG induction for 6 h at 37℃.It mainly existed in the form of inclusion body with the molecular weight 28 ku.Bioinformatics analysis showed that the 3C protein was an unstable and non-secretory protein, and it had no transmembrane regions but multiple phosphorylation sites.So it mainly involved in protein hydrolysis process.There was obvious differences in 3C gene among swKoV CH441 strain and reference strains of kobuvirus.As a common lyase of the small RNA virus, 3C protein played important roles in the processes of viral replication, transcription, translation and polyprotein maturation.This study successfully constructed the prokaryotic expression vector of 3C protein and predicted its structure, which provided a basis for the preparation of 3C protein crystals and the study of its crystal structure.

Key words: porcine kobuvirus; 3C gene; prokaryotic expression; protein structure prediction