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Monoclonal antibody ZK2C2 and application

A technology of antibodies and sequence lists, applied in the fields of application, antibodies, antiviral agents, etc., can solve the problems of increasing virus entry, aggravating the disease, and aggravating the severity of the disease, achieving significant application value and far-reaching social significance

Active Publication Date: 2018-11-02
GUANGZHOU EIGHTH PEOPLES HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that people who have been infected with different serotypes of dengue virus successively have the risk of aggravating the severity of the disease, that is, the antibodies produced by the pre-infected serotype of dengue virus can bind to the different serotypes of dengue virus reinfected, but cannot neutralize Viruses, on the contrary, increase the entry of viruses and aggravate the condition

Method used

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  • Monoclonal antibody ZK2C2 and application
  • Monoclonal antibody ZK2C2 and application
  • Monoclonal antibody ZK2C2 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the discovery of antibody

[0040] 1. Protein preparation

[0041] 1. Construction of recombinant plasmids

[0042] (1) Synthesize the double-stranded DNA molecule shown in Sequence 2 of the Sequence Listing.

[0043] The double-stranded DNA molecule shown in sequence 2 of the sequence listing encodes the protein shown in sequence 1 of the sequence listing, wherein the open reading frame is nucleotides 13-1296 from the 5' end of sequence 2.

[0044] In sequence 1 of the sequence listing, the 1st to 19th amino acid residues from the N-terminal form the signal peptide, the 20th to 421st amino acid residues form the E protein of Zika virus, and the 422nd to 427th amino acid residues form the His6 Label. Name the protein shown in Sequence 1 of the sequence listing as E-His 6 Fusion protein, expected molecular weight 50kDa.

[0045] (2) Double-digest the double-stranded DNA molecule obtained in step 1 with restriction endonucleases BamHI and NotI, and recov...

Embodiment 2

[0073] Example 2, Preparation of ZK2C2 Antibody

[0074] 1. Construction of recombinant plasmids

[0075] The double-stranded DNA molecule shown in sequence 4 of the sequence listing is inserted into the PMD18T vector to obtain a heavy chain expression vector. In sequence 4 of the sequence listing, nucleotides 1 to 888 form the CMV promoter, nucleotides 889 to 2280 encode the full-length heavy chain shown in sequence 3 of the sequence listing, nucleotides 2333 to 2478 for the ployA fragment.

[0076] The double-stranded DNA molecule shown in sequence 6 of the sequence listing is inserted into the PMD18T vector to obtain a light chain expression vector. In sequence 6 of the sequence listing, the 1st to 888th nucleotides constitute the CMV promoter, the 889th to 1596th nucleotides encode the full-length light chain shown in sequence 5 of the sequence listing, and the 1597th to 1742nd nucleotides for the ployA fragment.

[0077] 2. Antibody preparation

[0078] 1. The heavy ...

Embodiment 3

[0086] Example 3, ZK2C2 antibody neutralizing activity to virus

[0087] 1. Preparation of virus

[0088] Zika virus was inoculated into C6 / 36 cells (DMEM medium containing 10% FBS), placed at 28°C, 5% CO 2 Under the condition of static culture for 4 days, then centrifuged at 3000rpm for 5min, the supernatant was collected, which was the Zika virus liquid.

[0089] 2. Detection of neutralizing activity of monoclonal antibodies

[0090] 1. Take the ZK2C2 solution prepared in Example 2 and dilute it with PBS buffer (pH7.2, 10mM) to obtain antibody dilution.

[0091] 2. Inoculate Vero cells into a six-well plate (4 × 10 per well 5 cells), cultured overnight until the cell density reached 90% and evenly covered the well plate.

[0092]3. Mix the Zika virus solution prepared in step 1 with the antibody dilution prepared in step 1 to obtain each mixed solution (in each milliliter of mixed solution, the virus content is 100 pfu, and the antibody concentration is 40 μg / mL, 8 μg / mL...

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Abstract

The invention discloses a monoclonal antibody ZK2C2 and application. The invention provides an IgG antibody named as ZK2C2 and composed of light chains and heavy chains. CDR1, CDR2 and CDR3 in a heavychain variable region of the heavy chains are sequentially the No. 45-52 amino acid residues, No. 70-77 amino acid residues and No. 116-122 amino acid residues from N terminal in sequence 3 of a sequence table; CDR1, CDR2 and CDR3 in a light chain variable region of the light chains are sequentially the No. 45-52 amino acid residues, No. 70-72 amino acid residues and No. 109-119 amino acid residues from N terminal in sequence 5 of the sequence table. The invention further provides application of the IgG antibody in preparation of a medicine for inhibiting Zika virus. The antibody disclosed bythe invention has significant application value on prevention and control of Zika virus diseases and produces profound social significance.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody ZK2C2 and its application. Background technique [0002] Zika virus, dengue virus, West Nile virus and yellow fever virus belong to the flavivirus genus. Zika virus disease is a mosquito-borne infectious disease caused by Zika virus with mild or no symptoms. The main manifestations are mild fever, rash (mostly maculopapular), headache, arthralgia, muscle pain, weakness, and non-suppurative conjunctivitis. In recent years, the Zika virus has broken out in French Polynesia and Brazil successively, and continues to spread rapidly all over the world, especially in Latin America and the Caribbean, which has become an international public health emergency. [0003] Zika virus infection causes only mild symptoms. However, in the prevalence of Polynesia, adults have serious neurological complications - Guillain-Barré syndrome (GBS), with an incidence rate ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCA61K2039/505C07K16/1081C07K2317/565C07K2317/76Y02A50/30
Inventor 张复春庾蕾张林琦王若珂高菲
Owner GUANGZHOU EIGHTH PEOPLES HOSPITAL
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