隆林黑猪氨肽酶N基因的生物信息学分析和原核表达

本研究旨在进行猪氨肽酶N（porcine aminopeptidase N，pAPN）基因扩增和生物信息学分析，并在大肠杆菌中进行表达。从三元杂商品猪和广西地方猪隆林黑猪中扩增出pAPN基因，通过ExPASy和Mega 6.0等软件对其进行生物信息学和系统进化分析；将pAPN克隆到原核表达载体pET-32a（+），构建重组质粒pET-32a-pAPN，使其在大肠杆菌Rosetta（DE3）感受态细胞中表达。经IPTG诱导表达、纯化，产物用SDS-PAGE和Western blotting进行分析。结果显示，隆林黑猪和三元杂商品猪的pAPN基因长2 949 bp，开放性阅读框为2 892 bp，编码963个氨基酸，同源性为100%，与GenBank数据库中公布的标准序列（登录号：NM_214277）相比，隆林黑猪共有5处氨基酸突变，其中Phe82Asn、Leu107Phe、Leu108Ile和Ser330Pro 4处突变位于pAPN酶催化活性区域，Val621Ile突变位于APN病毒结合区域。pAPN为不稳定的亲水性蛋白，跨膜区位于第17~33位氨基酸，有12个N-糖基化位点，33个B细胞抗原表位预测位点，三级结构为同源二聚体。Val621Ile突变可能影响APN结合病毒的能力。试验成功构建重组质粒pET-32a-pAPN，SDS-PAGE鉴定以包涵体形式表达，纯化后的蛋白在123 ku处有明显条带，且具有良好的抗原性，为进一步获得多克隆抗体、研究该基因和TGEV、PEDV、PDCoV的关系奠定基础。

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

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.