全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

一种高效、稳定的可溶性原核表达载体的构建及应用

DOI: 10.7668/hbnxb.2009.06.009, PP. 46-49

Keywords: 口蹄疫病毒,原核表达,可溶性,信号肽

Full-Text   Cite this paper   Add to My Lib

Abstract:

以O型口蹄疫病毒结构蛋白VP1基因重组克隆载体pGEM-OVP1为模板,设计表达引物,经PCR扩增获得上游带有PelB信号肽编码序列的VP1C端编码区,将其克隆至该表达载体pET-30a(+)中,构建了可溶性原核表达载体pET-30a-PelB-VP1C.将VP1全长编码区替换VP1C,获得重组原核表达载体pET-30a-PelB-VP1.将两种重组质粒分别转化大肠杆菌工程菌株BL21-(DE3)-plysS,经IPTG诱导表达,实现了VP1全长及其C端的可溶性表达,以金属离子螯合层析法分别对表达的VP1及其C端融合蛋白进行纯化,SDS-PAGE显示纯化的目的蛋白分别在32ku和20ku处有单一目的条带,具有较高的纯度.Westernblot分析,VP1蛋白及其C端均可与牛O型口蹄疫病毒阳性血清反应.对重组菌株的连续传代实验证实了该可溶性表达载体的遗传稳定性,显示了该可溶性原核表达载体在蛋白可溶性表达中的应用价值.

References

[1]  Baneyx F.Recombinant protein expression in Escherichia coli[J].Curr Opin Biotechnol,1999,10(5):411-421.
[2]  Swartz J R.Advances in Escherichia coli production of therapeutic proteins[J].Curr Opin Biotechnol,2001,12(2):195-201.
[3]  Makrides S C.Strategies for achieving high-level expression of genes in Escherichia coli[J].Microbiol Rev,1996,60(3):512-538.
[4]  Baneyx F,Mujacic M.Recombinant protein folding and misfolding in Escherichia coli[J].Nat Biotechnol,2004,22(11):1399-1408.
[5]  Singh S M,Panda A K.Solubilization and refolding of bacterial inclusion body proteins[J].J Biosci Bioeng,2005,99(4):303-310.
[6]  Choi J H,Lee S Y.Secretory and extracellular production of recombinant proteins using Escherichia coli[J].Appl Microbiol Biotechnol,2004,64(5):625-635.
[7]  Lei S P,Lin H C,Wang S S,et al.Characterization of the Erwinia carotovora pelB gene and its product pectate lyase[J].J Bacteriol,1987,169(9):4379-4383.
[8]  周建华,丛国正,高闪电,等.FMDV OA/58病毒株VP1蛋白结构构建与B细胞抗原表位的预测[J].华北农学报,2007,22(4):176-179.
[9]  Pugsley A P.The complete general secretory pathway in gram-negative bacteria[J].Microbiol Rev,1993,57(1):50-108.
[10]  Mergulhāo F J,Summers D K,Monteiro G A.Recombinant protein secretion in Escherichia coli[J].Biotechnol Adv,2005,23(3):177-202.
[11]  Mori H,Ito K.The Sec protein-translocation pathway[J].Trends Microbiol,2001,9(10):494-500.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133