隆林黑猪氨肽酶N基因的生物信息学分析和原核表达

doi:10.16431/j.cnki.1671-7236.2020.06.002

Abstract

Abstract: The aim of this study was to amplify the porcine aminopeptidase N (pAPN) gene and analyze with bioinformatics,and express it in Escherichia coli.pAPN gene was amplified from three-way hybrid commercial pigs and Guangxi local Longlin Black pigs,and its bioinformatics and phylogenetic analysis were carried out by ExPASy and Mega 6.0 software.Then pAPN gene was cloned into prokaryotic expression vector pET-32a(+) to construct the recombinant plasmid pET-32a-pAPN,and expressed in Escherichia coli Rosetta(DE3).The expression was induced by IPTG,the product was analyzed by SDS-PAGE and Western blotting.The results showed that pAPN gene of Longlin Black pigs and three-way hybrid commercial pigs was 2 949 bp in length,the open reading frame was 2 892 bp,encoding 963 amino acids,and the homology was 100%.Compared with standard pAPN gene (accession No.:NM_214277) sequence in GenBank database,there were 5 amino acid mutations in Longlin Black pigs,of which Phe82Asn,Leu107Phe,Leu108Ile and Ser330Pro were located in the catalytic activity region of pAPN enzyme and Val621Ile mutation was located in APN virus combined area.PAPN was an unstable hydrophilic protein,the transmembrane region was between 17th and 33th amino acid.There were 12 N-glycosylation sites,33 B cell antigen epitope prediction sites,and the tertiary structure was homologous dimer.Val621Ile mutation might affect the ability of APN binding virus.The recombinant plasmid pET-32a-pAPN was successfully constructed and expressed in the form of inclusion body by SDS-PAGE.The purified protein had obvious band at 123 ku and had good antigenicity,which laid a foundation for further obtaining polyclonal antibody and studying the relationship between the gene and TGEV,PEDV and PDCoV.

Key words: pAPN gene; prokaryotic expression; variation site

CLC Number:

S813.3

YUAN Tingting, QIN Yibin, WANG Ruomu, LIU Fang, CHEN Ying, WEI Zuzhang, HE Ying, HUANG Weijian, ZHAO Wu, OUYANG Kang. Bioinformatics Analysis and Prokaryotic Expression of Porcine Aminopeptidase N Gene in Longlin Black Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1648-1658.

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Bioinformatics Analysis and Prokaryotic Expression of Porcine Aminopeptidase N Gene in Longlin Black Pigs

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