Human antibody specifically binding four serotype dengue viruses

A dengue virus and antibody technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulins, etc., can solve the problems of long half-life, high price, weak affinity, etc., and achieve the effect of preventing infection

Active Publication Date: 2019-06-25
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with mouse-derived antibodies, humanized antibodies have the following characteristics: (1) Immunogenicity is lower than that of mouse-derived antibodies; (2) Affinity is weaker than that of mouse-derived antibodies, and specificity is better than that of mouse-derived antibodies; (3) Humanized Antibody Fc segment can induce the effector function of the b

Method used

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  • Human antibody specifically binding four serotype dengue viruses
  • Human antibody specifically binding four serotype dengue viruses
  • Human antibody specifically binding four serotype dengue viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1, Preparation of Fully Human Monoclonal Antibody 6B1

[0050] Peripheral blood was collected from 3 patients who were clinically diagnosed as convalescent patients with dengue fever (patients gave informed consent), 10 mL per person, 30 mL in total. Isolate mononuclear lymphocytes, use flow cytometry to sort single B cells in two steps, first sort out CD19 high CD20 low to negative CD3 negative , in sorting out CD27 from high CD38 high B cells were divided into 96-well plates, one cell per well. Antibody light and heavy chain variable region genes were amplified from single B cells by reverse transcription PCR and nested PCR, and PCR products were sequenced for those with correct pairing of light and heavy chains, and multiple pairs of light and heavy chain variable region gene sequences including 6B1 were obtained. Among them, the heavy chain variable region gene of antibody 6B1 is shown in the 1-363 position from the 5' end of SEQ ID No.3; the light ch...

Embodiment 2

[0060] Example 2, the serotype specificity of monoclonal antibody 6B1

[0061] C6 / 36 cells were used to inoculate and culture four serotypes of dengue virus, and the culture supernatant was collected to harvest the virus stock solution. Take an appropriate amount of virus stock solution, add 4×Loading buffer, and boil in a water bath to prepare protein samples. 10 μL protein sample was taken for SDS-PAGE, and then the protein sample was electrotransferred to PVDF membrane (product of Amersham Company, catalog number 10600023), and blocked with 5% skimmed milk. Add the antibody to be tested 6B1 (1 μg / mL) and incubate at room temperature for 2 hours or overnight in a refrigerator at 4°C. Wash the membrane with TBS-T three times, 10 min each time, add 1:5000 diluted HRP-Goat anti human IgG (product of Zhongshan Jinqiao Company, Cat. No. ZB2304), and incubate at room temperature for 1 h. The membrane was washed three times with TBS-T, 10 min each time. Add developer solution to...

Embodiment 3

[0063] Example 3, ELISA detection of binding of monoclonal antibody 6B1 to antigen

[0064]The 96-well enzyme-linked plates were coated with four serotypes of dengue virus recombinant E protein (products of RayBiotech Company, product numbers are 228-11688, 228-11689, 228-11690, 228-11691), 2 μg / mL, 100 μL / wells, coated overnight at 4°C; or coated with four serotypes of inactivated dengue virus culture supernatants, 100 μL / well, coated overnight at 4°C. After discarding the coating solution, wash with PBST for 5 min / time x 3 times, add 2% BSA-PBST, 200 μL / well, and place at 37°C for blocking for 2 hours. Add 3-fold diluted 6B1 antibody (concentration: 30 μg / mL-0.17 ng / mL), 50 μL / well, and incubate at 37° C. for 2 h. Discard the primary antibody, wash with PBST for 5 min / time x 3 times, add 1:5000 diluted secondary antibody HRP-Goat anti human IgG (product of Zhongshan Jinqiao Company, Cat. No. ZB2304), and incubate at 37°C for 1 h. Discard the secondary antibody, wash with ...

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Abstract

The invention discloses a human antibody specifically binding four serotype dengue viruses. According to the human antibody, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in a heavy chain variable region are shown as the positions 26-33, the positions 51-58 and the positions 97-121 from the N end in the SEQ ID No.1. The amino acid sequences of LCDR1, LCDR2 and LHCDR3 in a light chain variableregion are shown as the positions 27-32, the positions 50-52 and the positions 89-97 from the N end in the SEQ ID No.2. The antibody can effectively prevent infection of four serotype DENVs, and treat the infection of lethal doses of DENV-1 and DENV-2. The antibody is of great significance for the development of drugs for preventing and treating dengue virus infection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human antibody specifically binding to four serotypes of dengue viruses. Background technique [0002] Dengue virus (DENV) belongs to the Flaviviridae family Flavivirus genus, is an enveloped single positive-sense RNA virus, according to the antigenicity of its envelope, it is divided into four serotypes (DENV I-IV). Dengue virus is mainly transmitted by Aedes aegypti and Aedes albopictus, and is widely prevalent in tropical and subtropical regions. The first human infection with dengue virus will cause dengue fever (Classic Dengue Fever, DF), which is a self-limiting febrile disease. At the same time, dengue virus can stimulate good cellular immunity and humoral immunity, which can provide life-long protection for this subtype of infection. immunity. However, when re-infected with other subtypes of dengue virus, the antibodies produced by the first infection will carry the virus...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12Q1/70A61K39/42A61P31/14
CPCA61K39/42A61P31/14C07K16/10C12Q1/70Y02A50/30
Inventor 杨志新王荣陆健昇余云舟周晓巍
Owner ACADEMY OF MILITARY MEDICAL SCI
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