Humanized antibody for resisting severe fever with thrombocytopenia syndrome bunyavirus (SFTSV)

A technology of cloning antibodies and monoclonal antibodies, which is applied in the direction of antiviral immunoglobulins, etc., can solve the problems of heterogeneity hindering the application, limited sources, mismatch of donor and recipient blood types, etc., and achieve the effect of strong clinical application and economic value

Inactive Publication Date: 2014-03-19
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sources of polyantibody plasma are not only limited, but its clinical application is also limited by conditions such as difficult quality control, mismatch of donor and recipient blood types, and potential infectious agents.
Mouse monoclonal antibody is easy to prepare and has a clear therapeutic mechanism, but its heterogeneity hinders its application in humans

Method used

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  • Humanized antibody for resisting severe fever with thrombocytopenia syndrome bunyavirus (SFTSV)
  • Humanized antibody for resisting severe fever with thrombocytopenia syndrome bunyavirus (SFTSV)
  • Humanized antibody for resisting severe fever with thrombocytopenia syndrome bunyavirus (SFTSV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Purification of JS-2010-003 virus particles

[0039] 1. Materials

[0040] Virus strain JS-2010-003: It was isolated by the patent applicant from the serum of a patient infected with SFTSV in Jiangsu in 2010.

[0041] 2. Methods and Results

[0042] After the virus was inoculated into Vero cells, at 37°C, 5% CO 2 Cultivate in the incubator for 10 days, aseptically absorb the virus culture supernatant; use 1:4000 diluted β-propiolactone (β-propiolactone) to inactivate at 4°C for 24h, and centrifuge at 5000rpm for 30min to remove cell debris; Centrifuge for 2 hours; resuspend the virus particles with a small amount of phosphate-buffered saline (PBS), and further purify the virus with a molecular sieve chromatography column Sepharose 4FF. JS-2010-003 virus particles with high purity were collected through the above steps. All virus handling was performed in a biosafety level 2 (BSL-2) laboratory.

Embodiment 2

[0043] Example 2 Construction and screening of phage display scFv human antibody library

[0044] 1. Materials

[0045] Primers:

[0046] V H Gene amplification primers:

[0047] Upstream primer: HSCVH1-F, see SEQ ID NO: 11 in the sequence listing; HSCVH2-F, see SEQ ID NO: 12 in the sequence listing; HSCVH35-F, see SEQ ID NO: 13 in the sequence listing; HSCVH3a-F, see SEQ ID NO: 13 in the sequence listing ID NO: 14, HSCVH4-F, see SEQ ID NO: 15 in the sequence listing; HSCVH4a-F, see SEQ ID NO: 16 in the sequence listing;

[0048] Downstream primer: HSCG1234-B, see SEQ ID NO: 17 in the sequence listing;

[0049] V κ Gene amplification primers:

[0050] Upstream primer: HSCK1-F, see SEQ ID NO: 18 in the sequence listing; HSCK24-F, see SEQ ID NO: 19 in the sequence listing; HSCK3-F, see SEQ ID NO: 20 in the sequence listing; HSCK5-F, see sequence SEQ ID NO: 21 in the list;

[0051] Downstream primer: HSCJK14o-B, see SEQ ID NO: 22 in the sequence listing; HSCJK2o-B, see SE...

Embodiment 3

[0073] Example 3 ScFv Antibody Neutralizing Activity Screening

[0074] 1. Materials

[0075] The SFTSV strains used in the experiments are listed in the table below.

[0076]

[0077] a The SFTS patients came from Anhui or Shandong, and the patients were admitted and the virus was isolated in medical institutions in Jiangsu Province.

[0078] 2. Methods and results

[0079] (1) Prokaryotic expression and purification of scFv antibody fragments. Transform the positive phage clone plasmid obtained in Example 2 into E. coli TOP10F' competent cells, spread Amp plates, pick a single clone, shake and culture at 37°C overnight; transfer the overnight bacterial solution 1:100 to fresh SB Shake culture medium at 37°C until OD600 is about 1.0; add IPTG with a final concentration of 1mmol / L, shake at 250rpm at 37°C for 16h; centrifuge at 12000rpm at 4°C for 20min, discard the supernatant, and use binding buffer ( Containing 20mmol / L PBS, 8mol / L urea, 20mmol / L imidazole) 500mL re...

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Abstract

The invention discloses a humanized antibody for resisting severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). A nucleotide sequence of the antibody is that a light chain nucleotide sequence is shown as SEQ ID NO.5, and a heavy chain nucleotide sequence is shown as SEQ ID NO.6. A method for preparing the antibody is disclosed. Specific recognition is performed on SFTSV viral particles through the antibody. Neutralization of the antibody on the SFTSV is utilized, the antibody is made into a specific antibody drug, and the specific antibody drug can be used for preventing and treating diseases caused by SFTSV infection clinically.

Description

technical field [0001] The present invention relates to an antibody, in particular to an anti-SFTSV human antibody. Background technique [0002] In 2010, a number of public health incidents caused by tick bites occurred in parts of central and eastern China, including Anhui, Jiangsu, Hubei, Henan, Shandong and other provinces, which caused social panic and seriously affected the health and safety of local people and economic development. . Through the joint research of many units including the national and relevant provincial CDCs, it was found that the pathogen causing the infectious disease is a new type of hemorrhagic fever virus, which belongs to the Bunyaviridae family in taxonomy, and is currently named fever Fever with thrombocytopenia associated with a novel bunyavirus in China.N Engl Med 2011;364 (16): 1523-1532]. With the strengthening of national monitoring, the virus has also been found in Zhejiang, my country. In addition, there have been recent reports of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10
Inventor 焦永军张黎曾晓燕郭喜玲史智扬崔仑标周明浩
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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