Severe fever with thrombocytopenia syndrome virus (SFTSV) inhibitor and application thereof

A technology of inhibitors, nucleic acid molecules, applied in the field of molecular immunology

Active Publication Date: 2019-11-12
JIANGSU PROVINCIAL CENT FOR DISEASE CONTROL & PREVENTION PUBLIC HEALTH RES INST OF JIANGSU PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no anti-SFTSV antibody on the market at home and abroad, so the establishment and development of antibody-based detection and diagnosis and treatment supplies with independent intellectual property rights has important practical significance for the intervention of various related diseases

Method used

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  • Severe fever with thrombocytopenia syndrome virus (SFTSV) inhibitor and application thereof
  • Severe fever with thrombocytopenia syndrome virus (SFTSV) inhibitor and application thereof
  • Severe fever with thrombocytopenia syndrome virus (SFTSV) inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Screening of anti-SFTSV-Gn protein single chain antibody

[0091] 1. Purification of JS-2010-014 virus particles

[0092] 1.1 Materials

[0093] JS-2010-014, obtained by the patent applicant in 2010 from the peripheral blood of a Jiangsu patient in the acute phase.

[0094] 1.2 Methods and results

[0095] After the virus was inoculated into Vero cells, at 37°C, 5% CO 2 Cultured for 5 days under certain conditions, the supernatant was aseptically collected and the 50% tissue culture infectious dose (50% tissue culture infective dose, TCID50) was measured. The virus suspension was inactivated by 1:4000β-propiolactone at 4°C for 24 hours, centrifuged at low speed to remove cell debris, suspended in PBS after ultracentrifugation for 2 hours, and further purified by molecular sieve chromatography. JS-2010-014 virus particles with high purity can be obtained through the above steps, and all virus operations are performed in a biosafety level 2 (BSL-2) laborator...

Embodiment 2s

[0156] Example 2 Full molecular construction and eukaryotic expression of scFv antibody

[0157] 1. Construction of baculovirus recombinant plasmid

[0158]Use 4-6, 2F6, and 1B2 single-chain antibody plasmids as templates to amplify VH and VL genes by PCR respectively. Each amplification system contains the following contents: scfv plasmid 0.1μg, upstream primer 60pmol, downstream primer 60pmol, 10× PCR buffer 10 μL, dNTP 8 μL, MgCl 2 6 μL, Ex Taq 0.5 μL, add water to 100 μL. The reaction conditions are: 94°C for 5 min; 94°C for 15 sec, 56°C for 30 sec, 72°C for 1 min, 30 cycles; 72°C for 10 min. The gel extraction kit was used to recover the target band. The above PCR products were digested with XhoI / NheI (VH) and SacI / HindIII (Vk) respectively, and the eukaryotic baculovirus expression vector plasmid pAc-K-CH3 was first digested with XhoI / NheI, and the VH fragment was inserted Afterwards, carry out SacI / HindIII digestion and insert the VL fragment. The enzyme digestion...

Embodiment 3

[0161] Example 3 Binding Experiment of Whole Molecular Antibody and SFTSV

[0162] 1. Method

[0163] (1) Inoculate Vero cells in a 24-well plate, culture at 37° C., and inoculate each well with virus liquid when the fusion rate reaches 90%.

[0164] (2) Two days later, remove the virus liquid, add 400 μl of 4% paraformaldehyde to each well, and fix at room temperature for 30 minutes.

[0165] (3) Discard the paraformaldehyde, wash with PBS three times, add 400 μl 0.2% Triton X-100 to permeabilize the cell membrane, and keep at room temperature for 15 minutes.

[0166] (4) Discard the Triton X-100, wash with PBS 3 times, add 5 mg / ml BSA for blocking, room temperature for 30 min.

[0167] (5) Discard the blocking solution, add 300 μl of purified whole-molecular monoclonal antibody to each well, set up duplicate wells, and set up a blank control group without adding antibodies and 4-5 IgG1 monoclonal antibody (see patent literature: a human anti-SFTSV Source antibody, authori...

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Abstract

The invention discloses a severe fever with thrombocytopenia syndrome virus (SFTSV) inhibitor. The SFTSV inhibitor is a monoclonal antibody against SFTSV, wherein variable structural domain of heavy chain of the SFTSV inhibitor contains the sequence of CDR-H1 given in SEQ ID NO:1, the sequence of CDR-H2 given in SEQ ID NO:2 and the sequence of CDR-H3 given in SEQ ID NO:3; and moreover, variable structural domain of light chain of the SFTSV inhibitor contains the sequence of CDR-L1 given in SEQ ID NO:4, the sequence of CDR-L2 given in SEQ ID NO:5 and the sequence of CDR-L3 given in SEQ ID NO:6.The invention further extends to therapeutic use of the SFTSV inhibitor, compositions thereof, and a method for detecting SFTSV thereby.

Description

technical field [0001] The invention belongs to the field of molecular immunology, and relates to an SFTSV inhibitor and application thereof. Background technique [0002] Fever with thrombocytopenia syndrome (severe fever with thrombocytopenia syndrome, SFTS) is a new type of tick-borne arboreal hemorrhagic fever. A new, natural foci, acute infectious disease caused by SFTSV infection, commonly known as "tick disease", since it was discovered in the central and eastern regions of my country in 2009, with the strengthening of monitoring, many Localities have reported confirmed cases one after another, and cases have also been reported from time to time in Japan, South Korea, the United States, and the United Arab Emirates. SFTS has posed a serious threat to global human health. [0003] Most members of the Bunyaviridae are transmitted by vector organisms, such as ticks, mites, mosquitoes, rats, etc., causing regional or global epidemics. In my country, mainly Hantavirus (Han...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/63G01N33/68A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/10C07K2317/565C12N15/63G01N33/6854
Inventor 张文帅郭喜玲焦永军秦祥荣于学杰曾晓燕迟莹施伟庆
Owner JIANGSU PROVINCIAL CENT FOR DISEASE CONTROL & PREVENTION PUBLIC HEALTH RES INST OF JIANGSU PROVINCE
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