Establishment of prokaryotic expression and purification as well as test method of reconstruction influenza A H1N1 virus nonstructural protein NS1

A non-structural protein and influenza virus technology, applied in the field of zoonotic disease research, can solve problems such as epidemics or large-scale spread of human influenza, and achieve the effects of low cost, high biological safety, and simple technology

Inactive Publication Date: 2009-12-16
CUSABIO TECH LLC
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Problems solved by technology

However, after the spread and mutation of birds and mammals, ...

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  • Establishment of prokaryotic expression and purification as well as test method of reconstruction influenza A H1N1 virus nonstructural protein NS1
  • Establishment of prokaryotic expression and purification as well as test method of reconstruction influenza A H1N1 virus nonstructural protein NS1
  • Establishment of prokaryotic expression and purification as well as test method of reconstruction influenza A H1N1 virus nonstructural protein NS1

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Embodiment 1

[0034] The construction of the prokaryotic expression vector of the nonstructural protein NS1 gene of embodiment 1 type A H1N1 influenza virus

[0035] Digest PUC57-NS1 and vector pGEX-6P-1 with BamHI and SalI, recover NS1 gene and vector pGEX-6P-1; then ligate with T4 DNA ligase, and transform E. coli Rossetta competent cells at 22°C for 1 hour, at 37°C For culture, randomly select multiple single colonies, place them in LB liquid medium and culture them at 37°C for 5 hours, add IPTG to a final concentration of 1mM when the OD value of the bacteria is about 0.6, and select positive clones after a small amount of expression identification. .

Embodiment 2

[0036]Example 2 A large amount of induced expression and protein purification of non-structural protein NS1 of influenza A (H1N1) virus

[0037] pGEX-6P-1-NS1 was expressed in Escherichia coli Rosetta, the clone was inoculated into 3ml LB buffer containing 100ug / ml ampicillin, 37°C, overnight, 220rpm; the overnight culture was diluted 1:100 to 1L containing 100ug / ml Ampicillin in LB buffer solution, 37°C, 220rpm, culture for 5h; add IPTG with a final concentration of 0.1mM, 22°C, 180rpm, continue to culture for 22h; centrifuge at 8000g, 15min, 4°C to harvest the bacteria; the cells were washed with 50mM Tris- Cl, 200mM NaCl, pH8.0, suspended to 25ml, added 5mg lysozyme, RT, 0.5h; sonicated with a sonicator until the solution was clear, 12000rpm, centrifuged 3 times for 20min; the supernatant was used on Glutathione-Sepharose4B column of GE healthcare The material was purified to obtain a purified fusion protein. The GST tag was digested by PSP, and the column material was pur...

Embodiment 3

[0038] Example 3 Type A H1N1 Infection Virus NS1-ELISA Differential Diagnosis Kit Composition

[0039] 1.1 Antigen-coated microwell plate (8 wells x 12 strips x 1 piece)

[0040] 1.2 HRP-labeled anti-human IgG 1 bottle (120ul / bottle)

[0041] 1.325 times washing liquid concentrate 1 bottle (20ml / bottle)

[0042] 1.4 Diluent 2 bottles (20ml / bottle)

[0043] 1 bottle of 1.5 substrate solution (10ml / bottle)

[0044] 1.6 1 bottle of stop solution (10ml / bottle)

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Abstract

The invention belongs to the research field of zoonosis, relating to a differential diagnosis method. Nonstructural protein NS1 gene in a disclosed influenza A H1N1 California virus strain (A/California/08/2009(H1N1)) cDNA sequence is taken as the research content, a gene synthesis method is used to obtain a gene fragment construction prokaryotic expression carrier Pgex-6P-1-NS1; positive recombinant plasmid is converted to colibacillus to obtain a recombination strain (Escherichia coli BL21 rosseta/Pgex-6p-1-NS1); the nonstructural protein NS1 of the purified H1N1 is obtained by a plurality of chromatography methods, and an enzyme linked immunosorbent assay differential diagnosis method is established in a manner that the expression protein obtains specificity through a immunization host animal specific to polyclonal or monoclonal antibody of the H1N1 nonstructural protein NS1, which is used for distinguishing patients suffering from influenza A H1N1 virus infection and other patients suffering from influenza virus infections.

Description

technical field [0001] The invention belongs to the field of research on zoonotic diseases and relates to a method for differential diagnosis of zoonotic diseases. The non-structural protein NS1 gene in the published A / California / 08 / 2009 (H1N1) cDNA sequence was selected as the research content, and the prokaryotic expression vector pGEX-6P- 1-NS1; the positive recombinant plasmid was transformed into Escherichia coli to obtain a recombinant strain (Escherichia coli BL21 rosseta / pGEX-6P-1-NS1); the purified non-structural protein NS1 of H1N1 was obtained by various chromatographic methods, and used The expressed protein obtains polyclonal or monoclonal antibodies specific for H1N1 non-structural protein NS1 by immunizing host animals, and establishes an enzyme-linked immunosorbent assay differential diagnosis method for distinguishing H1N1 influenza virus infection from other influenza virus infections of patients. Background technique [0002] H1N1 is a virus, a member of...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P21/02C07K14/11C07K1/16C07K16/10G01N33/543G01N33/536G01N33/53C12R1/19
Inventor 华权高
Owner CUSABIO TECH LLC
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