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Crystal of African swine fever virus (ASFV) DNA ligase, and preparation method and application of crystal

A DNA ligase, swine influenza virus technology, applied in the biological field, can solve problems such as low domain specificity

Inactive Publication Date: 2019-05-28
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Asfv PolX has the specificity as above, but due to Asfv The remaining domains of PolX have very low specificity compared to its family members

Method used

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  • Crystal of African swine fever virus (ASFV) DNA ligase, and preparation method and application of crystal
  • Crystal of African swine fever virus (ASFV) DNA ligase, and preparation method and application of crystal
  • Crystal of African swine fever virus (ASFV) DNA ligase, and preparation method and application of crystal

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation that is derived from the DNA ligase of African swine fever virus

[0035] The sequence of DNA ligase derived from African swine fever virus is shown as SEQ ID NO: 1, which was obtained from literature reports (Hammond J. et. al., Nucleic Acids Research, 1992, 20(11): 2667-2671). Its one-letter amino acid sequence is as follows:

[0036] MLNQFPGQYS NNIFCFPPIE SETKSGKKAS WIICVQVVQH NTIIPITDEM FSTDVKDAVA

[0037] EIFTKFFVEE GAVRISKMTR VTEGKNLGKK NATTVVHQAF KDALSKYNRH ARQKRGAHTN

[0038] RGMIPPMLVK YFNIIPKTFF EEETDPIVQR KRNGVRAVAC QQGDGCILLY SRTEKEFLGL

[0039] DNIKKELKQL YLFIDVRVYL DGELYLHRKP LQWIAGQANA KTDSSELHFY VFDCFWSDQL

[0040] QMPSNKRQQL LTNIFKQKED LTFIHQVENF SVKNVDEALR LKAQFIKEGY EGAIVRNANG

[0041] PYEPGYNNYH SAHLAKLKPL LDAEFILVDY TQGKKGKDLG AILWVCELPN KKRFVVTPKH

[0042] LTYADRYALF QKLTPALFKK HLYGKELTVE YAELSPKTGI PLQARAVGFR EPINVLEII

[0043] Using molecular cloning technology, the codon-optimized full-length gene of African s...

Embodiment 2

[0045] Embodiment 2, African swine fever virus DNA ligase activity verification

[0046] Use reaction system 100mM NaCl; 20mM Tris pH 8.0, 10mM MgCl 2 , 1mM ATP, 0.8μM FAM fluorescently labeled DNA, 0.05μM protein. Incubate the protein and the substrate at 37 degrees Celsius for 5 minutes, then add the protein to the substrate, sample according to the time gradient, and add stop buffer (90% formamide, 20mM EDTA, 0.05% bromophenol) to each sample at a ratio of 1:1 blue, 0.05% xylene blue) to terminate the reaction by heating at 95°C. The samples after the reaction were terminated were separated by 16% urea gel, scanned by Typhoon FLA 9000, and the results were quantitatively analyzed by ImageQuantTL and GraphPadPrism software.

Embodiment 3

[0047] Embodiment 3, the crystallization method of African swine fever virus DNA ligase

[0048]Concentrate the pre-prepared high-purity protein to 0.48mM, and add an equal volume of double-stranded DNA with a length of 22bp at a corresponding concentration of 0.5mM, using a crystallization kit (Crystal ScreenKit I / II from companies such as Hampton Research, Index, Salt, PEG / Ion, etc.) were used as the initial screening conditions for crystal growth, and the sessile drop meteorological diffusion method was used for crystallization. After optimization and adjustment, finally under the conditions of 0.1M Ammoniun acetate, 0.01 M BIS-TRIS pH 5.5, and 10% PEG1000 Crystals with high diffraction resolution can be obtained.

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a crystal of African swine fever virus (ASFV) DNA ligase, and a preparation method and application of the crystal.The DNA ligase AsfvLIG contains 419 amino acids, has the molecular weight of about 45 kDa, and has highly soluble expression in an expression strain Escherichia coli BL21 (DE3). A three-dimensional structure of the crystal is obtained by X-ray diffraction, with the resolution reaching 2.5 angstroms. A crystallographic asymmetric unit has a protein molecule consisting of 9 alpha helixes and 17 betapleated sheets, and is combined with DNA of a catalytic reaction substrate. The 153th asparagine, the 211th leucine, the 402th leucine and the 403th glutamine in the structure have different conformations from human DNA ligase. The crystal structure of the DNA ligase AsfvLIG is widely used in drug design or drug screening for African swine fever viruses (ASFV).

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to African swine influenza virus Asfivirus DNA ligase Asfv LIG expression purification, crystal growth method, crystal structure and its application. Background technique [0002] African swine fever virus (ASFV) is a double-stranded DNA virus belonging to Asfivirus genus, and is currently the only known Asfarviridae family virus. The ASFV virus is very contagious and can infect both wild and domestic pigs. The incubation period of the ASFV virus is very short, and in some cases symptoms will appear in about two days. The early symptoms of African swine flu include fever, anorexia, and general weakness in limbs, etc. These are similar to the symptoms of normal swine flu, so early African swine flu is often misdiagnosed as normal swine flu. In the later stage, pigs infected with ASFV will have symptoms different from normal swine flu, such as hypothermia and red skin, but at this...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00G16C20/50
Inventor 甘建华李继喜陈屹勍
Owner FUDAN UNIV
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