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Mature human beta-defensin-2 (HBD-2) and preparation method thereof

A β-defensin, a mature technology, applied in the field of gene recombination expression and genetic engineering, can solve the problems that the purity of mature HBD-2 cannot be guaranteed, it is not suitable for large-scale preparation, and the health of experimenters is unfavorable, so as to achieve stable expression, convenient source, The effect of reducing production costs

Active Publication Date: 2013-05-08
SOUTH CHINA UNIV OF TECH
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Problems solved by technology

Although the yield and purity of HBD-2 in the cell-free expression system are high, its preparation cost is too high and it is not suitable for large-scale preparation; the treatment time is relatively long to remove the propeptide of HBD-2 by enterokinase treatment, generally It takes 8-16 hours, the cleavage efficiency is relatively low, and enterokinase also has a certain degree of non-specific cleavage (S.H.Shahravan, X.Qu, I.S.Chan, J.A.Shin, Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag.Protein Expression and Purification59, 2008, 314-319); and the removal of HBD-2 propeptide and enterokinase by ion exchange chromatography cannot guarantee the purity of mature HBD-2; the use of cyanogen bromide cleavage method to remove HBD-2 pre Peptide, which has strong toxicity, is not good for the health of experimenters, and is not conducive to the later application, and there is also a certain non-specific cleavage

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  • Mature human beta-defensin-2 (HBD-2) and preparation method thereof
  • Mature human beta-defensin-2 (HBD-2) and preparation method thereof
  • Mature human beta-defensin-2 (HBD-2) and preparation method thereof

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Embodiment 1

[0040] 1. Construction of engineered bacteria producing recombinant human β defensin-2 prepropeptide

[0041] By introducing a glutamate residue between the encoding human β defensin-2 propeptide and the mature sequence as a recognition site for glutamate-specific endopeptidase, and fusing a histidine tag at the N-terminus, introducing a termination at the C-terminus After being digested with NdeI and XhoI, it is connected to the expression vector pET22b(+) to realize the construction of the recombinant plasmid pET22b-HBD-2 prepropeptide. The specific construction process is as follows: figure 1 Shown.

[0042] 1. Design and synthesis of target gene fragments and primers

[0043] According to the human β defensin-2 gene sequence (GenBank ID: AY155577), GAA was introduced as a glutamate codon between the mature sequence gene and its propeptide gene fragment, and the new recombinant HBD-2 prepropeptide gene sequence (see SEQ ID NO: 3) was synthesized by Shanghai Jierui Biological Engi...

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Abstract

The invention discloses mature human beta-defensin-2 (HBD-2) and a preparation method thereof. The method comprises the following steps of: introducing glutamic residue between mature sequence and propeptide of HBD-2, obtaining HBD-2 pre-pro-peptide through recombination expression, obtaining HBD-2 pre-pro-peptide with higher purity via nickel column affinity chromatography purification, carrying out enzymolysis by using recombined bacillus licheniformis glutamic specific endopeptidase with an His(histidine)-tag, removing the propeptide and the affinity tag and removing the recombined bacillus licheniformis glutamic specific endopeptidase and other impurities through the affinity chromatography again, wherein the mature HBD-2 with higher purity exists in the solution. The preparation method of the mature HBD-2, disclosed by the invention, has the advantages of high efficiency, low cost and simplicity in purification process and accordingly lays a foundation for the large-scale preparation and the application of the mature HBD-2.

Description

Technical field [0001] The invention belongs to the technical field of gene recombination expression in the field of genetic engineering, and specifically relates to a novel preparation method of mature human beta defensin-2. Background technique [0002] Human beta defensin (HBD) is a cationic antimicrobial peptide rich in cysteine, which has a strong killing effect on gram-negative bacteria, gram-positive bacteria, fungi and even viruses. It is widely used in cosmetics and antiseptic fields. Human beta defensin-2 (HBD-2) was found in psoriasis skin lesions in 1997 (J. Harder, J. Bartels, E. Christophers, J. Schroder, A peptide antibiotic from human skin. Nature, 1997 ,(387):861-861), is one of the members of the HBD family. Its cationic region can interact with the anionic phospholipid head gene and water molecules of the bacterial cell membrane to form electrostatic force and form voltage-dependent pores in the membrane. Inserted into the membrane to form multimers to form l...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/12C07K14/47C07K1/22
Inventor 王菊芳叶伟王海鹰马毅于平儒王小宁
Owner SOUTH CHINA UNIV OF TECH
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