绵羊NYD-SP27基因的原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2020.03.001

摘要/Abstract

摘要： 本研究旨在对绵羊NYD-SP27基因进行克隆和原核表达，并对其表达的蛋白进行生物信息学分析。根据GenBank中绵羊NYD-SP27基因序列（登录号：KX905090）设计1对特异性引物，通过PCR方法扩增目的基因片段，构建pET-22b（+）-NYDSP27重组质粒并转化E.coli DH5α感受态细胞，提质粒进行双酶切鉴定，将鉴定正确的pET-22b（+）-NYDSP27重组质粒转化E.coli BL21（DE3）感受态细胞，经IPTG诱导表达，对融合蛋白进行SDS-PAGE及Western blotting鉴定，并利用生物信息学方法对NYD-SP27基因编码的氨基酸序列进行分析。结果显示，试验成功扩增出大小为1 617 bp的NYD-SP27基因片段，并经NdeⅠ和Xho Ⅰ双酶切获得大小为5 400和1 617 bp的两条片段，表明成功构建了pET-22b（+）-NYDSP27重组质粒，诱导表达重组蛋白大小约60 ku，主要以包涵体的形式存在。NYD-SP27蛋白分子式为C₂₇₉₈H₄₃₁₉N₇₃₇O₈₁₉S₁₉，原子总数为6 892，理论等电点（pI）为6.16，为酸性蛋白，不稳定系数为46.96，属于不稳定蛋白，总平均亲水性为-0.403。该蛋白无跨膜结构，无信号肽，含有45个潜在的磷酸化位点和24个抗原表位；NYD-SP27蛋白二级结构中的α-螺旋、β-折叠、延伸链和无规则卷曲分别占26.21%、3.90%、18.03%和51.86%。本试验结果可为深入研究绵羊NYD-SP27蛋白的作用机理提供理论依据。

关键词: NYD-SP27基因; 原核表达; 生物信息学分析

Abstract: This study was aimed to clone and express the NYD-SP27 gene in sheep,and analyze the bioinformatics of its expressed protein.A pair of specific primers were designed based on the sequence of NYD-SP27 gene (accession No.:KX905090) in GenBank,and the NYD-SP27 gene fragment was amplified by PCR method.The recombinant plasmid pMD19-T-NYDSP27 was constructed and then transformed into E.coli DH5α competent cells.The plasmid was identified by restriction enzyme digestion.The recombinant plasmid pET-22b(+)-NYDSP27 was constructed and then transformed into E.coli BL21(DE3) competent cells.The expressed protein was induced by IPTG and identified by SDS-PAGE and Western blotting.The amino acid sequence encoded by NYD-SP27 gene was analyzed by bioinformatics methods.The results showed that the 1 617 bp gene fragment was successfully amplified,and two fragments of 5 400 and 1 617 bp were digested by Nde Ⅰ and Xho Ⅰ,indicating that the recombinant plasmid pET-22b(+)-NYDSP27 was successfully constructed.The expressed recombinant protein was about 60 ku.The NYD-SP27 recombinant protein was an inclusion body with a molecular formula of C₂₇₉₈H₄₃₁₉N₇₃₇O₈₁₉S₁₉.The total number of atoms was 6 892,the theoretical isoelectric point (pI) was 6.16,which was an acidic protein,the instability coefficient was 46.96,which was an unstable protein,and the average hydrophilicity was -0.403.The protein had no signal peptide and transmembrane structure,and had 39 potential phosphorylation sites and 24 epitopes.In the secondary structure of NYD-SP27 protein,alpha helix,beta turn,extended chain and random coil accounted for 26.21%,3.90%,18.03% and 51.86%,respectively.This results might provide reference data and material basis for further study of regulatory mechanisms of NYD-SP27 protein in sheep.

Key words: NYD-SP27 gene; prokaryotic expression; bioinformatics analysis

中图分类号:

李娜, 吴雨红, 袁利明, 张晓晓, 赛务加甫. 绵羊NYD-SP27基因的原核表达及生物信息学分析[J]. 中国畜牧兽医, 2020, 47(3): 645-654.

LI Na, WU Yuhong, YUAN Liming, ZHANG Xiaoxiao, Saiwujafu. Prokaryotic Expression and Bioinformatics Analysis of NYD-SP27 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 645-654.

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