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Fully humanized trivalent specific antibody for neutralizing tetanus toxin and preparation method thereof

A tetanus toxoid, fully humanized technology, applied in the field of genetic engineering, can solve the problems of no clone expression of the sequence, no production application value, and little antibody expression, etc., so as to reduce production costs and reduce human allergy. , the effect of significant economic and social benefits

Active Publication Date: 2020-11-20
XIAMEN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] With the hybridoma monoclonal technology and phage display technology, people have gradually used these technologies to conduct research in this field, but there are still some problems in these researches: ① The screened sequences have not been cloned and expressed, only sequence analysis has been done; ② The cloned and expressed sequences have only been subjected to in vitro ELLISA and / or in vitro binding experiments, and the protective effect in vivo is unknown; ③ After some sequences have been expressed, neutralization and protection experiments in mice have been carried out. A common idea is a variety of monoclonal antibodies Mixing is similar to the "cocktail" method. There are two options for this idea. One is to express the monoclonal antibodies separately and then mix them; the other is to express the monoclonal antibodies in a mixed manner.
The former is more than twice the production cost of recombinant monoclonal antibody, and has no production application value
The latter has the disadvantages of low product purity and low yield, resulting in high cost
[0005] Pichia pastoris is currently the most commonly used protein expression system next to Escherichia coli, and has been recognized by the US FDA as a GRAS (Generally recognized as safe) microorganism. It has characteristics that other systems do not have, such as methanol utilization, high-density fermentation, There are few by-products, etc., but Pichia pastoris can seriously o-glycosylate the protein, which leads to the immune response of the human body, so it is rarely used for antibody expression

Method used

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  • Fully humanized trivalent specific antibody for neutralizing tetanus toxin and preparation method thereof
  • Fully humanized trivalent specific antibody for neutralizing tetanus toxin and preparation method thereof
  • Fully humanized trivalent specific antibody for neutralizing tetanus toxin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Recombinant expression and purification of glycosidase.

[0018] 1.1 Construction of glycosidase expression sequence plasmid

[0019] The nucleotide sequence (SEQ ID NO: 1) of a-1,2mannosidase (Genebank: XP_006962575) of Trichoderma reesei, encoding 526 amino acids, was biosynthesized by Shanghai Jierui, and then MscI was added to the 5 end of the sequence Restriction site and 3 ends add XhoI restriction site, these sequences are loaded into pET20a vector at last and are transformed in Escherichia coli BL21 (DE) 3, the product obtained after expression is named 6*His-TrMannl (sequence 1 , SEQ ID NO: 1), because it contains Trx and 6*His tags, its molecular weight is about 67KD.

[0020] SEQ ID NO: 1 Trichoderma reesei a-1,2mannosidase (Genebank: XP_006962575) nucleotide sequence (Genebank: XM_006962513), CATATG boxed at the 5-end is NdeI restriction site, CTCGAG boxed at the 3-end is XhoI restriction site location.

[0021] AGATTCCCTAGCAGCTCCGTCCTTGCCCTCGG...

Embodiment 2

[0026] Example 2 Obtaining of Fully Humanized Sequence

[0027] 2.1 Acquisition of ScFv sequence

[0028] According to the previous research of the applicant, after optimization, the following optimized human sequences (A sequence, B sequence and C sequence) were obtained, wherein, these sequences specially optimized by the applicant are suitable for expression in Pichia pastoris, And does not contain the sequence of EcoRI, SalI, BglII and NotI restriction enzyme cutting sites.

[0029] A sequence

[0030] A sequence heavy chain variable region protein sequence (A-H-P sequence, SEQ ID NO: 2)

[0031] EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIY YASWVKGRFTISRDNAKNSLYLQXNSLRAEDTALYYCAKDIGYCTGGVCPQA

[0032] A sequence light chain variable region protein sequence (A-L-P sequence, SEQ ID NO: 3)

[0033] QFYADSAPLCVGVSGEDGNHLLHPQQWQHCQQLCAVVPTAPGQFPTTVIYEDHQRPSG IPDRFSASIDSSSSNSASLIISGLKTEDEADYYCQSYDTNNRVFGGGTKLTVLG

[0034] A sequence heavy chain variable regio...

Embodiment 4

[0091] The measurement results showed that A, B and C had neutralizing and protective effects respectively, and their titers were 200IU / mg, 150IU / mg and 120IU / mg respectively. The combination has a synergistic neutralizing protective effect: ①The potency of group A+B is 350IU / mg; ②The potency of group B+C is 280IU / mg; ③The group has the best protective effect, and its titer is higher than the current horse anti-tetanus F(ab)2 titer 400-450IU / mg (because the molecular weight of horse anti-tetanus F(ab)2 is small) and human tetanus 200IU / mg of Influenza Immunoglobulin (TIG). Therefore, in the next experiments, the A+B+2C trivalent antibody was further expressed.

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Abstract

The invention provides a fully humanized trivalent specific antibody for neutralizing tetanus toxoid. The trivalent specific antibody is composed of a single chain I and a single chain II, each singlechain comprises two single-chain variable fragments (scFv), a human hinge region of IgG1 and an Fc fragment, and the four single-chain variable fragments (scFv) are composed of respectively-named A,B and C fragments and can respectively play a neutralizing role. The trivalent specific antibody is expressed in the same pichia pastoris strain, glycosidase is used for treating a purified product, amouse neutralization experiment shows that the antibody has a protective effect, and the antibody has a pharmaceutical effect and can be used as a diagnostic reagent.

Description

technical field [0001] The invention belongs to the technical field of biotechnology related to genetic engineering. More specifically, the invention discloses a fully humanized neutralizing anti-tetanus toxin trivalent specific antibody and a preparation method thereof. Background technique [0002] Tetanus is a specific infection caused by Bacillus tetani invading wounds, and its main pathogen is the tetanus toxoid produced by Bacillus tetani. Tetanus toxoid is an approximately 150 kDa protein consisting of 1315 amino acids. After the proteolytic enzyme of Bacillus tetani, the protein is cleaved between 456 and 467 amino acid residues into components of about 50KD light chain (LC) and 100KD heavy chain (LN), LC and LN pass through a pair of disulfide bonds S439-S467 connection. There is a papain cleavage site between 864 and 863 that can further degrade the heavy chain into N-terminal (HCN) and C-terminal (HCC or TTC). HCN is responsible for passage of synaptic vesicles...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/81G01N33/569C12R1/84
CPCC07K16/1282C07K16/468C07K16/461C12N15/815G01N33/56911C07K2317/31C07K2317/24C07K2317/622C07K2317/40C07K2317/35G01N2333/33
Inventor 李海浪曾凡伟
Owner XIAMEN MEDICAL COLLEGE
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