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Xinjiang hemorrhagic fever virus nucleoprotein antigen gene as well as recombinant protein and application thereof

A technology of hemorrhagic fever virus and antigen gene, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2010-12-22
THE CENT FOR DISEASE CONTROL & PREVENTION OF XINJIANG UYGUR AUTONOMOUS REGION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently, there is no effective prevention and treatment for CCHF

Method used

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  • Xinjiang hemorrhagic fever virus nucleoprotein antigen gene as well as recombinant protein and application thereof
  • Xinjiang hemorrhagic fever virus nucleoprotein antigen gene as well as recombinant protein and application thereof
  • Xinjiang hemorrhagic fever virus nucleoprotein antigen gene as well as recombinant protein and application thereof

Examples

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Effect test

Embodiment 1

[0019] Example 1: The Xinjiang hemorrhagic fever virus nucleoprotein N235 antigen gene of the present invention was obtained in large quantities by using polymerase chain reaction (PCR) technology and gene recombination technology.

[0020] Primers were designed according to the S gene sequence of Xinjiang hemorrhagic fever virus, and the target fragment was amplified by PCR using cDNA as a template. The PCR reaction system is: cDNA 2μL, 10×Ex Taq Buffer 2μL, Sterilized dH 2 O 12.9 μL, Ex Taq (5U / μL) 0.5 μL, P1primer (20 μM) 0.3 μL, P2primer (20 μM) 0.3 μL, dNTP Mixture (each 10 mM) 2 μL.

[0021] According to the nucleoprotein gene sequence of Xinjiang hemorrhagic fever virus, primers were designed.

[0022] Upstream primer P1 (Primer):

[0023] 5'-ccggatcccttgccaagcttgcagagactgaagggaagggagtg-3',

[0024] Downstream primer P2 (Primer):

[0025] 5'-ccgaattcctatcaatctgtgcaccctgtgcacgaagtgcagacgagtttttgt-3'.

[0026] The reaction conditions for PCR amplification of the XHFN...

Embodiment 2

[0027] Example 2: Obtaining the XHFNP235 recombinant protein of the present invention.

[0028] The XHFNP235 gene fragment of the present invention is cloned into the pGEX-KG prokaryotic expression vector, transformed into Escherichia coli BL21 (with expression function), inoculated in 3 mL LB medium containing ampicillin (50 μg / mL), 37 ° C, 220 rpm After shaking culture overnight, inoculate in LB medium containing the same concentration of ampicillin at 1:100, 37°C, 220rpm / min, culture for 3h, until OD 600 When ≈0.4, add IPTG to a final concentration of 0.4mmol / L, continue shaking culture for 3h, centrifuge at 4°C, 4000rpm / min for 12min, and collect bacteria. Suspend the cells with an appropriate amount of PBS, add loading buffer [0.08M Tris / HCl (pH 6.8), 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.005% bromophenol blue], boil for 10min, 12000× Centrifuge at g for 10 minutes, take 5 μL supernatant for 12% SDS-PAGE electrophoresis, and stain with Coomassie Brilliant Blue R-...

Embodiment 3

[0029] Example 3: Using the XHFNP235 recombinant protein of the present invention as an antigen, the ELISA method is used to detect animal serum samples collected from Xinjiang hemorrhagic fever endemic areas.

[0030] 3.1 Using XHFNP235 recombinant protein as antigen to establish ELISA detection method for XHFV antibody

[0031] (1) Antigen plate preparation: Dilute XHFNP235 recombinant protein to 1 μg / ml with coating solution (0.05M carbonate buffer solution at pH 9.6), 0.2ml per well of the enzyme plate, place in a 37°C incubator for 2 hours and transfer to Refrigerate at 4°C overnight, rinse with PBST 5 times the next day, and pat dry for later use.

[0032] (2) Add 100 μl of 1 / 100 diluted goat serum to each well of the antigen plate, put the ELISA plate in a humid box at 37°C for 1 hour, and do positive, negative, and blank controls at the same time; wash 4-5 times with PBST lotion Then pat dry, add 1 / 500 diluted rabbit anti-goat IgG-HRP enzyme marker 100μl, put it in a ...

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Abstract

The invention relates to the technical filed of antigen genes, in particular to a Xinjiang hemorrhagic fever virus nucleoprotein antigen gene (XHFNP235) as well as recombinant protein and application thereof. The gene has a nucleotide sequence as sequence 1. In the invention, the Xinjiang hemorrhagic fever virus nucleoprotein antigen gene (XHFNP235) and the recombinant protein thereof are obtained from Xinjiang hemorrhagic fever virus. Through PCR amplification, cloning, transformation, expression plasmid construction, induction expression and immunoassay detection, the Xinjiang hemorrhagic fever epitope is positioned at the 235th to 305th amino acid region of NP protein, and the region is a highly conservative amino acid region of the NP protein, which further shows that the XHFNP235 recombinant protein can be a candidate diagnosis antigen for Xinjiang hemorrhagic fever, and provides a new way for diagnosing and applying Xinjiang hemorrhagic fever.

Description

1. Technical field [0001] The invention relates to the technical field of antigen genes, and relates to a nucleoprotein antigen gene (XHFNP235) of Xinjiang hemorrhagic fever virus and its recombinant protein and application. 2. Background technology [0002] Crimean-Congo Hemorrhagic Fever (CCHF) is a viral natural foci disease that is widely prevalent in Africa, Asia, Eastern Europe and the Middle East, and its pathogen is Crimean Sub-Congo hemorrhagic fever virus (Crimean-Congo Hemorrhagic Fever Virus, CCHFV), the average case fatality rate is 10%-50%. [0003] Crimean-Congo hemorrhagic fever is also known as Xinjiang Hemorrhagic Fever (XHF) in my country. The pathogen Xinjiang Hemorrhagic Fever Virus (XHFV) is currently the only confirmed natural outbreak in my country. Biosafety Level 4 (Laboratory Biosafety Level 4, BSL-4) pathogens. [0004] Because the virus can be transmitted from person to person, the incidence of the virus is rare, and clinicians have no experienc...

Claims

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Application Information

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IPC IPC(8): C12N15/40C07K14/175G01N33/569
Inventor 张渝疆魏鹏飞孙素荣
Owner THE CENT FOR DISEASE CONTROL & PREVENTION OF XINJIANG UYGUR AUTONOMOUS REGION
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