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Expression and purification of amino terminal region of SARS coronavirus non-structural protein nsp2, and crystalline structure of amino terminal region

A non-structural protein, coronavirus technology, applied in the field of three-dimensional crystal structure, can solve the problems of inability to carry out, irreplaceable, and lack of experimental models for SARS coronavirus live virus experiments

Inactive Publication Date: 2013-06-26
TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, due to the strict restrictions on severe acute respiratory syndrome SARS coronavirus experiments in China and the lack of related experimental models, experiments on the live virus of SARS coronavirus cannot be carried out.
The next best thing is to study the mouse hepatitis virus MHV (mouse hepatitis virus) which belongs to the same species as the SARS coronavirus at the cellular level or the host animal model, or the pseudovirus or replicon of the SARS coronavirus, but these cannot Alternative to live SARS coronavirus

Method used

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  • Expression and purification of amino terminal region of SARS coronavirus non-structural protein nsp2, and crystalline structure of amino terminal region
  • Expression and purification of amino terminal region of SARS coronavirus non-structural protein nsp2, and crystalline structure of amino terminal region
  • Expression and purification of amino terminal region of SARS coronavirus non-structural protein nsp2, and crystalline structure of amino terminal region

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Experimental program
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Effect test

Embodiment 1

[0090] Transformation of SARS coronavirus non-structural protein nsp2 for expression in Escherichia coli

[0091] The inventor initially tried to express the full-length non-structural protein nsp2 protein. After trying on different expression vectors pGEX, pET, etc., and different expression strains, such as BL21(DE3), BL21 plys, etc., no stable protein could be obtained. . It is implied that the gene of SARS coronavirus non-structural protein nsp2 needs to be modified at the genetic level. Through the design of PCR amplification primers, starting from the first amino acid at the amino terminal of the SARS coronavirus non-structural protein nsp2, more than 4 clones that deleted amino acids one by one were constructed. The clone that maintains protein stability at 5°C for more than two weeks is SARS nsp2Met1-Arg277.

[0092] Expression and purification of SARS coronavirus nonstructural protein nsp2 in Escherichia coli

[0093] The SARS coronavirus non-structural protein nsp...

Embodiment 2

[0095] Crystallization of nonstructural protein nsp2 of SARS coronavirus

[0096] The non-structural protein nsp2 of SARS coronavirus expressed and purified by the above method is concentrated to a concentration of about 10 mg / ml, and is screened with a crystallization reagent (Screen Kit I / II, Index, etc. from companies such as Hampton Research) by the gas-phase hanging drop method Crystal growth conditions. After preliminary screening, the inventors obtained initial crystals under the conditions of 0.2M magnesium acetate tetrahydrate, 0.1M sodium cacodylate trihydrate (pH 6.5), and 20% (w / v) polyethylene glycol (PEG) 8,000 crystallization reagent. Through further optimization, wherein, under the condition of using 4% (v / v) n-propanol as additive, crystals with good appearance were obtained (see attached image 3 ). X-ray diffraction was carried out, and the parent crystal with a resolution of about 1.6 angstroms was selected, and the corresponding data were collected.

Embodiment 3

[0098]Crystallographic Diffraction Data Collection and Structural Analysis of SARS-CoV Nonstructural Protein nsp2

[0099] At the BL17A line station of the Japan High Energy Synchrotron Radiation Accelerator Agency, the wavelength is 1.0000 angstroms, and the detector is ADSC Q270. Figure 4 ), and then using the synchrotron radiation instrument located at the Shanghai Synchrotron Light Source BL17U-MX line station, under the X-ray wavelength at the absorption edge of the selenium element (0.9798 Å), the detector collects a set of diffraction resolution for ADSC Q315 with a resolution of 2.5 Å Diffraction data for single-wavelength anomalous scattering of . Using HK2000 (Otwinowski 1997) to process the data, it is found that the crystal has the P2(1)2(1)2 space group, and the unit cell parameters are about: a=67.1 angstroms, b=76.8 angstroms, c=65.0 angstroms, α=β=γ =90°. Through the calculation of Matthews constant, it is found that an asymmetric protein should contain 1 pr...

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Abstract

The invention provides a crystalline three-dimensional structure of an amino terminal region of an SARS (severe acute respiratory syndrome) coronavirus non-structural protein nsp2. The amino terminal region of the SARS coronavirus non-structural protein nsp2 comprises amino acids at positions in a range of about 1-50 to about 150-300 of the SARS coronavirus non-structural protein nsp2; and atoms in the crystalline three-dimensional structure have at least 40% of atomic coordinates in a table 1, or the average root mean square deviation of the main-chain carbon-skeleton atomic structure coordinates of the at least 40% amino acids of the crystalline three-dimensional structure of the amino terminal region of the SARS coronavirus non-structural protein nsp2 and the coordinates in the table 1 is not greater than 1.5 angstroms. The invention also provides an expression, purification and crystallization method of the SARS coronavirus non-structural protein nsp2, the crystalline structure of the SARS coronavirus non-structural protein nsp2, and theoretic guiding meanings and applications of the crystalline structure in functional researches, latent drug screening and drug designs.

Description

technical field [0001] The present invention relates to a method for genetically engineering severe acute respiratory syndrome SARS coronavirus nonstructural protein nsp2 to obtain high-efficiency expression, purification and crystallization in Escherichia coli, and severe acute respiratory syndrome SARS coronavirus nonstructural protein nsp2 Three-dimensional crystal structure of amino acids from position 1 to position 277. Background technique [0002] From the end of 2002 to the beginning of 2003, the large-scale outbreak of SARS in the world caused panic among all mankind, greatly challenged the ability of my country and the world to deal with major epidemics, and caused governments and scientists of various countries to attach great importance to the coronavirus. At present, although the large-scale outbreak of the SARS epidemic has been brought under control, there have been small-scale outbreaks after 2003. Although these small-scale outbreaks were monitored and cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/70
Inventor 饶子和李元元杨诚刘祥王权陈帅王敬兰张颖陈卫强
Owner TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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