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黄杆菌肝素酶I基因的克隆与表达

Cloning and Expression of Flavobacterium heparinum Heparinase I Gene
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摘要 目的克隆并表达黄杆菌肝素酶I(HepI)基因。方法通过PCR从黄杆菌基因组DNA中扩增黄杆菌HepI基因,验证后插入到表达质粒pET-22b中构建表达载体,重组质粒转化大肠杆菌BL21(DE3)进行蛋白质表达,并用Azure A法测定酶活性。结果PCR扩增得到序列正确的HepI基因,并将该基因成功插入到pET-22b表达载体中。重组质粒转入E.coli BL21(DE3)后表达的蛋白质经SDS-PAGE分析,与目的蛋白质的相对分子质量基本一致,其活性约为50 U/LA600。结论克隆并成功表达了黄杆菌HepI基因。 Objective To clone and express the heparinase Ⅰgene from Flavobacterium heparinum. Methods The heparinase Ⅰ gene was amplified with PCR from the genomic DNA of Flavobacterium heparinum, and it was linked into the expression plasmid pET-22b after sequencing, then, the recombinant plasmid was expressed in E.coli BL21 (DE3) and the enzyme activity of recombinant heparinase Ⅰ was verified by Azure A assay. Results The recombi- nant pET-22b with the PCR product of heparinase I which was confirmed by DNA sequencing was successfully constructed. The expression product of recombinant plasmid pET-22b in E.coli BL21 (DE3) was tested by SDSPAGE electrophoresis. The enzyme activity of recombinant heparinase Ⅰ was 50 U/LA600. Conclusion The heparinase Ⅰ gene from Flavobacterium heparinum can be cloned and expressed successfully.
出处 《食品与药品》 CAS 2008年第4期5-7,共3页 Food and Drug
关键词 肝素酶Ⅰ 克隆 基因表达 酶活性 heparinase Ⅰ clone genetic expression enzyme activity
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参考文献4

  • 1Kjellen L, Lindahl U. Proteoglycans: Structures and interactions[J]. Annu Rev Biochem, 1991, 60: 443-475.
  • 2Bernfield M, Gotte M, Park P W, et al. Functions of cell surface heparan sulkfate proteoglycans [J]. Annu Rev Biochem, 1999, 68: 729-736.
  • 3Iozzo R V. Matrix protegoglycan: from molecular design to cellular function [J]. Annu Rev Biochem, 1998, 67: 609- 615.
  • 4Galliher P M, Cooney R, Langer R J, et al. Heparinase production by Flavobacterium heparinum [J]. Appl Environ Microbiol, 1981, 41: 360-365.

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