Anti-Ebola virus monoclonal antibody, its preparation method and use

A monoclonal antibody, Ebola virus technology, applied in the fields of immunology and molecular biology, to achieve high ADCC activity, good antigen binding activity and virus neutralization activity, and good clinical application prospects.

Active Publication Date: 2021-07-16
BEIJING MABWORKS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, objective factors such as nearly 20,000 Chinese citizens working and living in affected areas in Africa, and the large number of business and trade exchanges between China and Africa have brought huge challenges to my country's prevention and control of the Ebola epidemic.

Method used

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  • Anti-Ebola virus monoclonal antibody, its preparation method and use
  • Anti-Ebola virus monoclonal antibody, its preparation method and use
  • Anti-Ebola virus monoclonal antibody, its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1: Antibody MIL77 nucleotide sequence obtained

[0128] The antibody amino acid sequence is recombinant antibody sequence, from Patent WO2009 / 094755 A1 and US2004 / 0053865A1. The reverse translation was selected in mammalian cells in mammalian cells, and the nucleotide sequence of the antibody MIL77 of the present invention was obtained by rational optimization of bioinformatics techniques, using molecular modeling and molecular biological mutation experiment.

[0129] Among them, the sequence of light chain nucleotides of the antibody MIL77-1 is:

[0130] GacatccagatgactcagtctccccccccctatctgtatctgtggggagaActGtctCccatcacatg Tcgagcaagtgagaatttacagtagttagcatgtatcagcagaaacagggaaaatctcctcagctcctgtc Tattctgcaacaatcttagcagatgtgtgccatcaaggtcagtggcagtgatcaggcactcagtattccccca AgatcaacagcctgcAgtctGaAgatttgggctTttctTctGtcaAcatttgggggTCCGTCGTTCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTCGTCCCGTTTTGGGGTCCCCGTTTTGGGGTACTCCCGTCGTTCGGGGGG AggggGgaccaagctggaaataaaaa cgtacggtggctgcacca...

Embodiment 2

[0153] Example 2: Cloning of antibody MIL77-1, MIL77-2 and MIL77-3 gene

[0154] Experimental material

[0155] PHUSION polymerase, TAQ DNA polymerase, restriction endonuclease, PGEM-T-Easy vector, Pyrobest Dnapolymese is NEB company products;

[0156] DNTP, DNA Marker standard is TAKARA products;

[0157] DNA Recycling Kit is QIAGEN's products;

[0158] TRANS2-Blue feels the full gold company product;

[0159]Small-to-medium plasmid kit (DP107-02) is the product of Tiangen biochemical company;

[0160] The large triump kit (DP117) is the product of the Tiangen biochemical company;

[0161] OPD is Sigma products;

[0162] Fitc marked sheep anti-human antibody: for PIERCE products;

[0163] Primer design is Biosun software;

[0164] Bimget Synthesis (GENEWIZ), Jin Weizhi Biotechnology (Beijing) Co., Ltd.

[0165] Gene sequencing is done by Beijing Noris Genetory Research Center Co., Ltd.

[0166] 2. Experimental methods and results

[0167] The lightweight chain variable region ge...

Embodiment 3

[0193] Example 3: Preparation of MIL77-1, MIL77-2 and MIL77-3 antibodies

[0194] Experimental material

[0195] The dry powder medium for entrusted Hyclone processing was prepared, and the cells were used to cultivate cells during the main cells, cell line screening and antibody preparation process, and the cell screening was added to the configured medium (Methionine Sulfoximine) (Methionine Sulfoximine) from SIGMA , MSX), used to prepare a cell count after formulating a solution from Sigma.

[0196] 2. Experimental methods and results

[0197] 2.1 Construction of antibody eukaryotic expression vector

[0198] The expression vector PTGS-FRT-DHFR (patent authorization number: ZL2005100643335.0) obtained by the Applicant is selected, and the hygromycin selection label is removed, and GS (Glutamine Synthetase, Glutamine Synthetase) is added to the PSHA1 and XHO1. Expressing the box as a screening mark; where GS cDNA is obtained from the cell line CHO expression GS through RT-PCR. ...

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PUM

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Abstract

The invention belongs to the fields of immunology and molecular biology, and relates to an anti-Ebola virus monoclonal antibody, its preparation method and application. Specifically, when it comes to anti-Ebola virus monoclonal antibodies or antigen-binding parts thereof, the amino acid sequences of the light chain and heavy chain variable regions are selected from a group of (1)-(3) as follows: (1 ) The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6; (2) the amino acid sequence of the light chain variable region is shown in SEQ ID NO As shown in: 10, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12; (3) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16, the amino acid sequence of the heavy chain variable region As shown in SEQ ID NO:18. The anti-Ebola virus monoclonal antibody of the invention has enhanced ADCC activity, good antigen binding activity and virus neutralization activity.

Description

Technical field [0001] The present invention belongs to the field of immunology, molecular biology, involving a monoclonal antibody against an Ebola virus, and methods and applications thereof. Background technique [0002] Ebola Virus is translated as an Ibola virus. It is a very rare virus. In 1976, it was discovered in the southern part of Sudan and the Ebola River in the southern Congolese (Golden) (Old Werald). Wide attention and attention, "Ebola" is here. Ebola virus is a strong infectious disease virus that can cause Ebola bleeding in human and primate, biosafety grade is 4 (AIDS is 3, SARS is Level 3, and the larger level is more Strictly); Ebola virus is mainly transmitted through paths of blood, saliva, sweat and secretions, and the virus incubation period can reach 2 to 21 days, but usually only 5 days to 10 days; the patients with viral infections mortality at 50 % To 90%; the cause of death is mainly stroke, myocardial infarction, low blood volume shock or multiple ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCC07K16/10C07K2317/92C07K2317/732C12N15/85C12N5/0682A61P31/14C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/14C07K2317/76C12N2800/107C12N2510/02
Inventor 李锋张伯彦叶培刘慧芳刘旭谢波赵健
Owner BEIJING MABWORKS BIOTECH
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