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Monoclonal antibody resisting EV-D68 virus and preparation and application thereof

A technology of EV-D68 and monoclonal antibody, which is applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., and can solve problems such as the difference in activity between antibodies and natural antibodies

Inactive Publication Date: 2018-11-13
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these technologies still have disadvantages: the gene pairing of light and heavy chains of antibodies produced is an unnatural random pairing, and the activity of the produced antibodies is different from that of natural antibodies

Method used

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  • Monoclonal antibody resisting EV-D68 virus and preparation and application thereof
  • Monoclonal antibody resisting EV-D68 virus and preparation and application thereof
  • Monoclonal antibody resisting EV-D68 virus and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: The preparation process of the monoclonal antibody against EV-D68 virus is described in detail.

[0040] Infection of rhesus monkeys with EV-D68 virus

[0041] 1) All animal experiments conform to the 3R principle, namely replacement (Replacement), reduction (reduction), optimization (Refinement); animal experiments are approved by IACUC (Institutional Animal Care and Use Committee), Institute of Medical Biology, Chinese Academy of Medical Sciences. Two healthy 6-month-old rhesus monkeys, weighing 1.2kg±0.3kg, were injected with EV-D68 virus with a titer of 104.5CCID50 via upper respiratory tract nasal drip. Before the experiment, the EV-D68 virus antibody test of the two rhesus monkeys was negative.

[0042] 2) At 1, 3, 5, 7, 9, 11, and 14 days after rhesus monkey infection, samples were taken to detect feces, nasal swabs, and blood viral load; at 0, 7, 14, 21, and 28 Daily detection of neutralizing antibody levels;

[0043] 3) Analysis of the results:...

Embodiment 2

[0210] Example 2: The monoclonal antibody A6-1 obtained in Example 1 was used to identify EV-D68 virus.

[0211] WB verification of antibody specificity

[0212] 1) Sample preparation: Add 80 μl of purified EV-D68 whole virus to 20 μl of 5× loading buffer, mix well and cook at 100°C for 5 minutes;

[0213] 2) Carry out SDS-PAGE gel protein electrophoresis of EV-D68 whole virus;

[0214] 3) After electrophoresis, take out the gel, cut off the stacking gel, and soak the separating gel in the transfer buffer; prepare a PVDF membrane of the same size as the gel, 2 filter papers, and soak in the transfer buffer; Stack neatly in the order of filter paper, PVDF membrane, gel, and filter paper, try to drive away the air bubbles during the stacking process, cover the negative plate, 25V, 1.3A and transfer the membrane for 20 minutes;

[0215] 4) Sealing: After the membrane transfer is completed, take out the PVDF membrane, put it into a glass plate, add 40ml of freshly prepared block...

Embodiment 3

[0219] Example 3: The monoclonal antibody A6-1 obtained in Example 1 was used to inhibit EV-D68 virus infection.

[0220] 1. Virus contact inhibition experiment

[0221] 1) Hela and Vero cells in the logarithmic growth phase were used, after trypsinization, the complete cell culture medium was used for pipetting and mixing, and the dilution was 4×10 5 cells / ml, add to 12-well cell culture plate according to 500 μl / well, and wait for the cells to grow into a monolayer for later use;

[0222] 2) Neutralize the virus with different dilutions of monoclonal antibodies, add the neutralized virus-antibody mixture to a 96-well cell culture plate grown into a single layer, react at room temperature for 1 hour, and wash the plate 3 times with PBS remove excess virus;

[0223] 3) Harvest the cells in the above 12-well plate, extract the total RNA of the cells, and perform quantitative detection of the virus gene copy number by qRT-PCR.

[0224] 4) Analysis of the results: From the res...

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Abstract

The invention discloses a monoclonal antibody resisting EV-D68 virus and preparation and application thereof. A single B cell separation technique is firstly adopted to conduct separation of the specific single memory B cell on a rhesus monkey infected by EV-D68 virus, paired antibody light and heavy chain genes are obtained, and the EV-D68 specific monoclonal antibody and the neutralizing activity can be obtained through further screening. The monoclonal antibody comprises a heavy chain and a light chain, and the nucleotide coding sequence and the amino acid coding sequence of the monoclonalantibody are as shown in the sequence graph. The invention provides an alternative drug for preventing and curing infection of EV-D68, and provides a new tool and new idea for identifying the EV-D68 virus.

Description

technical field [0001] The invention belongs to the technical field of preparation of anti-infective monoclonal antibodies, in particular to a monoclonal antibody capable of effectively inhibiting EV-D68 virus infection. At the same time, the present invention also relates to the preparation method of the monoclonal antibody, and the application of the antibody in EV-D68 virus identification, preparation of anti-infection drugs and the like. Background technique [0002] EV-D68 (Enterovirus D68, EV-D68) virus belongs to the Picornaviridae family and the Enterovirus genus. In 1962, it was isolated for the first time in the United States. In 2014, a large-scale outbreak and widespread prevalence of EV-D68 occurred in the United States and Canada. Subsequently, the epidemic of respiratory diseases caused by EV-D68 virus also appeared in Europe, Thailand, China, New Zealand and other regions. The EV-D68 virus can not only cause severe respiratory diseases, such as pneumonia, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/577G01N33/569A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/1009C07K2317/56G01N33/56983G01N33/577G01N2333/015
Inventor 刘龙丁孙明郑惠文郭磊王晶晶李冰香李恒杨泽宁李洪哲范海涛储曼曼黄星李楠杨瑾溪吴琼雯
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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