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Fully human monoclonal antibody against tetanus toxin and derivative thereof, and preparation method and application thereof

A monoclonal antibody, anti-tetanus technology, applied in biochemical equipment and methods, botanical equipment and methods, microorganism-based methods, etc., can solve problems that are limited to the laboratory stage and have not yet seen clinical applications, etc. , to achieve the effect of eliminating allergic reactions, prolonging the half-life in the body, and eliminating virus pollution

Active Publication Date: 2015-12-16
ANTAGEN BEIJING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, international and domestic research on anti-tetanus genetically engineered antibodies is limited to the laboratory stage, and no clinical application report has been seen

Method used

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  • Fully human monoclonal antibody against tetanus toxin and derivative thereof, and preparation method and application thereof
  • Fully human monoclonal antibody against tetanus toxin and derivative thereof, and preparation method and application thereof
  • Fully human monoclonal antibody against tetanus toxin and derivative thereof, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1 Preparation of anti-tetanus toxin monoclonal antibody hybridoma cell line

[0088] 5 x 10 cells from healthy blood donors immunized with tetanus toxoid vaccine were purchased from a commercial company (www.AllCells.com). 8 Peripheral blood mononuclear cells (trade name LeukoPak). Then on ice, the cells were incubated with anti-CD19-FITC and anti-CD27-PE antibodies (purchased from eBioscience) for 20 minutes, and after the cells were washed twice with PBS containing 1% FBS, the AriaII (BD Biosciences) flow cytometer Screen out CD19+CD27+ memory B cells. At the same time, Sp2 / 0-Agl4 mouse myeloma cells (purchased from ATCC, USA) in logarithmic growth phase in RPM1640+10% FBS culture medium were taken, washed three times with serum-free RPM1640 culture medium, and counted for later use. The memory B cells sorted above were mixed with Sp2 / 0 cells at a ratio of 10:1, and centrifuged at 1500 rpm for 7 minutes. Wash away the supernatant and prepare for fusion...

Embodiment 2

[0093] Example 2 Cloning of anti-tetanus toxin monoclonal antibody full-length heavy chain, light chain gene and variable region gene thereof

[0094] First, the total RNA of the tetanus toxin-resistant hybridoma (3B10) obtained in Example 1 was extracted using Qiagen's RNeasyPlusMini kit. then use IIFirstStrandcDNASynthesis Kit (NewEnglandBiolabs) used Oligo(dT)18 as a primer to reverse transcribe mRNA into cDNA. Using this cDNA as a template, design and synthesize degenerate primers (where W=A / T, K=G / T, R=A / G, Y=C / T, M =A / C, S=C / G, N=C / G / T, V=A / C / G).

[0095] The following are the upstream and downstream primers used to clone the antibody heavy chain and light chain genes respectively in Example 2:

[0096] Heavy chain upstream primer SEQ ID NO: 11

[0097] 5'-TGATCAGSACTGMACACAGAGRACTCACCATG-3'

[0098] Heavy chain downstream primer SEQ ID NO: 12

[0099] 5'-CTGACTCGAGTCATTTACCCGGAGACAGGGAGAGG-3'

[0100] Light chain upstream primer SEQ ID NO: 13

[0101] 5'-ATGGA...

Embodiment 3

[0128] Example 3 Construction of Anti-Tetanus Toxin Monoclonal Antibody L-P2A-H Form and ScFv-IgG1 Form Expression Vector

[0129] The L-P2A-H format is an antibody light chain (L) and heavy chain (H) linked by a self-cleaving P2A peptide. Its construction adopts the method of overlappingPCR. First, in the same PCR reaction system as above, the light chain portion was amplified with the following primers:

[0130] Anti-TTlightUPNheI.primer:

[0131] SEQ ID NO: 195'-GTGGGCTAGCGACTACCAGATGACCCAGTCTCC-3'

[0132] P2A-TTlightantisense.primer:

[0133] SEQ ID NO: 205'-CCGGCCTGCTTCAGCAGGCTGAAGTTGGTGGCTCCGCTGCCACACTCTCCCCTGTTGAAG-3'

[0134] The heavy chain portion was amplified with the following primers:

[0135] P2A-TT Theavysense.primer:

[0136] SEQ ID NO: 215'-CTGCTGAAGCAGGCCGGCGATGTGGAGGAGAATCCTGGCCCCATGGAGTTTGGGCTGAGC-3'

[0137] Anti-TTheavyASXhoI.primer:

[0138] SEQ ID NO: 225'-CTGACTCGAGTCATTTACCCGGAGACAGGGAGAGG-3'

[0139] The 750bp light chain and the 1.5kb h...

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Abstract

The invention provides a fully human monoclonal antibody against tetanus toxin. The amino acid sequences of a heavy-chain variable region and a light-chain variable region of the monoclonal antibody are SEQ ID No. 3 and SEQ ID No. 4, respectively; preferably, the amino acid sequences of a heavy chain and a light chain of the monoclonal antibody are SEQ ID No. 1 and SEQ ID No. 2, respectively. The invention further provides a preparation method and application of the fully human monoclonal antibody against tetanus toxin and a derivative thereof. The fully human monoclonal antibody against tetanus toxin provided by the invention can eliminate the biological risks of anaphylactic reaction and virus contamination, has a sufficiently long half life and high titer and in-vivo activity, can be applied to large-scale industrial production.

Description

technical field [0001] The invention belongs to the technical field of biological immunity, and in particular relates to a gene recombined fully human monoclonal antibody for neutralizing tetanus toxin. Background technique [0002] Tetanus, caused by Clostridium tetani infection, is a common and serious zoonotic disease with a high mortality rate. Clostridium tetani widely exists in soil, dust and rust, etc., and is also found in the digestive tract and feces of various animals, and the carrier rate in the crowd can reach up to 25%. Clostridium tetani can easily infect the human body through trauma, such as general trauma, trauma of women during childbirth, etc. After a person is infected by Clostridium tetani, during the incubation period of about seven days, Clostridium tetani grows and reproduces in a large number at the wound under anaerobic conditions and releases toxins. Tetanus toxin invades the central nervous system by destroying human nerve cells, leading to gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/85C12N5/20A61K39/40A61P31/04C12R1/91
Inventor 高闻达岳国华段富刚杨思仪
Owner ANTAGEN BEIJING BIOTECH CO LTD
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