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Antibody against varicella-zoster virus

A technology of herpes zoster virus and chickenpox, applied in the direction of antiviral agent, antiviral immunoglobulin, antibody, etc., can solve the problem of inability to eliminate the complications of VZV infection, achieve no xenogeneic serum reaction, strong specificity and good affinity Effect

Active Publication Date: 2021-05-11
ZHUHAI TRINOMAB BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although existing vaccines and systemic antiviral drugs can prevent and control VZV infection, they cannot eliminate numerous complications of VZV infection

Method used

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  • Antibody against varicella-zoster virus
  • Antibody against varicella-zoster virus
  • Antibody against varicella-zoster virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 Plasma Cell Sorting

[0103] Volunteers were inoculated with varicella vaccine (Merck, Zostavax) according to the manufacturer's method, and blood samples were collected on the 7th day after inoculation, and plasma and PBMC cells were separated by density centrifugation. For specific methods, please refer to the invention patent with the authorized announcement number CN107760690B . The VZV gH / gL protein complex (CAMBRIDGEBIO, Cat No: 01-11-0045) was selected as the antigen for serum antibody titer ELISA detection, and the sample with the highest fold change in antibody titer was screened out (dilution 50 times, OD value >2.0) for flow sorting. By flow cytometry, single plasma cells were sorted by CD3 / CD14 / CD16 / cd235a-CD19+CD20+ / -CD38hi CD27hi gating, and the specific plasma cell population was isolated, and the fully human monoclonal anti-VZV was isolated from them For the gene sequence of the antibody, please refer to the invention patent with the authoriz...

Embodiment 2

[0104] Example 2 Isolation of the variable region gene of the antibody of interest

[0105] The first strand of cDNA was synthesized by reverse transcription from the plasma cells obtained in Example 1 using constant region primers (see primer information disclosed in CN107760690B) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). The antibody gene was then isolated by the following PCR procedure: the first round of PCR: 50ul system contained 5ul of reverse transcription reaction product, 5 units of Taq enzyme, 0.2mM dNTPs, and 0.5uM of heavy chain and light chain of each subtype Antibody constant region primer (sequence), reaction conditions: pre-denaturation at 95°C for 5min, then 35 PCR cycles, each cycle: 95°C×30s, 55°C×60s, 72°C×90s, and finally extended at 72°C for 7min . The second round of PCR: 50ul system contains 2.5ul of the first round of PCR reaction product, 5 units of Taq Plus enzyme, 0.2mM dNTPs, and 0.5uM of heavy chain and light chain ant...

Embodiment 3

[0107] Embodiment 3 constructs the expression of recombinant antibody

[0108] The obtained PCR product of antibody variable region gene was connected to the pcDNA3.3 vector containing human IgG1 constant region by TA cloning method to construct the expression vector of fully human neutralizing antibody against varicella-zoster virus, and then The expression vector was transformed into DH5α competent bacteria for vector amplification and recombinant plasmid was extracted. Co-transfect HEK293 cells with the obtained recombinant plasmid and the transfection reagent PolyFect, 37°C, 8% CO 2 Cultured in an incubator, the paired heavy chain and light chain gene expression vectors were expressed in the cells, the supernatant was collected after 96 hours of culture, the cell debris was discarded by centrifugation, and the supernatant was purified by Protein A affinity chromatography. The purified antibody was tested by SDS-PAGE, and the results were as follows: figure 1 It shows tha...

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Abstract

The invention relates to a neutralizing monoclonal antibody or an antigen-binding fragment thereof, which are specific for varicella-zoster virus and bind with high affinity, and a preparation method of producing the antibody. The antibody has high potency in neutralizing varicella-zoster virus infection. The invention further relates to an epitope to which the antibody bind, and an application of the antibody in diagnosis, prevention and treatment of infected individuals.

Description

Background technique [0001] Varicella-zoster virus (VZV) belongs to human herpes simplex virus and is an enveloped virus. VZV has a high degree of species specificity, and its natural infection only occurs in humans and gorillas. The envelope of VZV contains various glycoproteins, such as gE, gB, gH, gI, gC, gL and so on. VZV glycoproteins are not only involved in viral entry into cells, but can also be transferred from infected to uninfected cells, which can elicit humoral and cellular immune responses to the virus. [0002] When VZV is first infected, it enters the body through the conjunctiva and respiratory mucosa to cause chickenpox, that is, the primary infection of varicella (varicella), which is a highly contagious common disease in children. In healthy children, chickenpox is a self-limiting disease with a duration of about 4 to 5 days; in immunocompromised children and newborns with no immunity, chickenpox infection may cause serious complications, such as viral pne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13A61K39/42A61P31/22G01N33/577G01N33/569
CPCC07K16/088A61P31/22G01N33/577G01N33/56994A61K2039/505C07K2317/565C07K2317/92G01N2333/04G01N2469/20C07K2317/21C07K2317/76
Inventor 廖化新王月明郑伟宏李嘉祺
Owner ZHUHAI TRINOMAB BIOTECHNOLOGY CO LTD
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