CN111961136B

CN111961136B - Fully humanized trivalent specific antibody for neutralizing tetanus toxin and preparation method thereof

Info

Publication number: CN111961136B
Application number: CN202010669181.2A
Authority: CN
Inventors: 李海浪; 曾凡伟
Original assignee: Xiamen Medical College
Current assignee: Xiamen Medical College
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2022-07-08
Anticipated expiration: 2040-07-13
Also published as: CN111961136A

Abstract

The invention provides a fully humanized trivalent specific antibody with the function of neutralizing tetanus toxoid, which consists of single chains I and II, wherein each single chain comprises 2 single-chain variable fragments (scFv), a hinge region of 1 human IgG1 and 1Fc fragment, and the four single-chain variable fragments (scFv) consist of two C fragments which are respectively named as A, B and can respectively play a neutralizing role. The trivalent specific antibody is expressed in the same strain of pichia pastoris, and a purified product is treated by glycosidase, mouse neutralization experiments show that the trivalent specific antibody has a protection effect, and the antibody has pharmaceutical and diagnostic reagent purposes.

Description

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

Technical Field

The invention belongs to the technical field of biotechnology and genetic engineering, and particularly discloses a fully humanized trivalent specific antibody for neutralizing anti-tetanus toxin and a preparation method thereof.

Background

Tetanus is a specific infection caused by invasion of a wound by tetanus bacillus, and the main pathogen is tetanus toxoid produced by tetanus bacillus. Tetanus toxoid is a protein consisting of 1315 amino acids and having a molecular weight of approximately 150 kDa. This protein is cleaved between 456 and 467 amino acid residues into approximately 50kD Light Chain (LC) and 100kD heavy chain (LN) components following the action of tetanus bacterium proteolytic enzymes, and the LC and LN are linked by a pair of disulfide bonds S439-S467. A papain (papin) cleavage site between 864 and 863 allows further degradation of the heavy chain to the N-terminus (HCN) and C-terminus (HCC or TTC). HCN is responsible for synaptic vesicles passing through the cytoplasm into the cytoplasmic membrane, while TTC is responsible for toxin binding to neuronal cells. In the TTC segment, two regions function as receptors for binding neurons, i.e., a W pocket with Trp1289, His1271, and Try1290 as cores, and an R pocket with Asp1147, Arg1226, Asn1216, Asp1214, and Try1229 as core regions.

Tetanus treatmentThe most effective treatment is the injection of Tetanus Antitoxin (TAT), horse tetanus immunoglobulin F (ab)₂Or human Tetanus Immunoglobulin (TIG). Tetanus Antitoxin (TAT) and horse tetanus immunoglobulin are antitoxins prepared by immunizing horses with tetanus toxoid, and have different forms, wherein the antitoxin in the two forms is derived from horses and has a certain anaphylactic reaction, and human Tetanus Immunoglobulin (TIG) is an antitoxin prepared by immunizing human bodies with tetanus toxoid, has the defects of no anaphylaxis, high cost, low source, complex preparation, insufficient supply and the like. TAT is still widely used clinically in China, and TIG is used only under the condition that TAT allergy test is positive or the initiative of family members requires. It is desirable to find a fully human specific antibody that is non-allergenic and inexpensive, and that can replace current antibody sources.

With the advent of hybridoma monoclonal technology and phage display technology, research has been conducted in this field using these technologies, but these studies have still presented several problems: firstly, the sequence screened is not subjected to clone expression, and only sequence analysis is carried out; secondly, the sequence of the cloning expression is only subjected to in vitro ELLISA and (or) in vitro combination experiments, and the in vivo protection effect is not known; thirdly, after some sequences are expressed, a mouse in-vivo neutralization protection experiment is carried out, one common idea is that a plurality of monoclonal antibodies are mixed in a mode similar to a cocktail, and the idea scheme has two schemes, namely, monoclonal antibodies are respectively expressed and then mixed; one is mixed expression of monoclonal antibody. The former is more than twice the production cost of the recombinant monoclonal antibody and has no production and application value. The latter has the disadvantages of low product purity and low yield, resulting in high costs.

Pichia pastoris is currently the most common protein expression system second only to Escherichia coli, is recognized as GRAS (generally recognized as safe) microorganism by the FDA in the United states, and has the characteristics that other systems do not have, such as methanol utilization, high-density fermentation, few byproducts and the like, but Pichia pastoris can cause severe o-glycosylation of protein, so that the protein is caused to cause immune response of a human body, and therefore the Pichia pastoris is rarely used for antibody expression.

Disclosure of Invention

One of the technical problems to be solved by the invention is to provide a fully humanized trivalent specific antibody against tetanus, which has the function of neutralizing tetanus toxin.

One of the technical problems to be solved by the present invention is to provide a method for mass production of trivalent specific antibodies with low price and low immunity.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one technical scheme, A, B and C three ScFV (single-stranded variable fragment) sequences are obtained in the previous work, and have a neutralization effect after being expressed in Pichia pastoris, and further a mixed method is adopted for detection, and the result shows that the mixed ScFV has a synergistic protection effect.

In a second aspect of the invention, the trivalent specific antibody is constructed and expressed recombinantly. First, the above sequence was ligated with the hinge region and Fc region of human IgG1 to construct pPIC₉In the k vector, specific antibodies of the ScFV-hinge-Fc type were formed, and the CH3 region of FC was mutated so that two chains form a knob-in-hole (see FIG. 2). Secondly, pPIC containing I and II chains₉And after the k vector is linearized by SalI and BglII restriction enzymes respectively, the linearized vector is introduced into Pichia pastoris GS115 in an electrotransfer mode, so that two sites of His gene and AOX1 are formed on GS115 chromosome and integrated at fixed points, and the expression product forms a double chain of a specific antibody. Finally, the antibody is expressed recombinantly, and the product is obtained by methods such as cation chromatography.

A third aspect of the invention is the further processing of the specific antibody. Because the glycosylation of the pichia pastoris product is O-type, the pichia pastoris product is easy to cause human immune reaction, and the product uniformity is low, the O-sugar chain is removed by adopting an in vitro enzyme digestion method. Firstly, glycosidase (a-1, 2 manosidase of Trichoderma reesei) is expressed in a recombination way, glycosylation of a trivalent specific antibody expressed in the Pichia pastoris in a recombination way is cut off, an expression product is purified, and the titer of the product is measured by a mouse neutralization experiment and can reach 550 IU/mg.

The invention has the beneficial effects that: obtain fully humanized doubleCompared with a tetanus antitoxin, the specific antibody overcomes the heterogeneity, and reduces the human body anaphylaxis by adopting glycosidase enzyme to remove O-glycosylation peculiar to pichia pastoris; tetanus immunoglobulin F (ab')₂The method overcomes the heterogeneity, reduces the anaphylaxis of human body, and can prolong the half-life period in vivo. Compared with human Tetanus Immunoglobulin (TIG), the method overcomes the defects of limited source and high production cost. Compared with the scheme of 'cocktail' adopting a plurality of monoclonal antibodies for mixing, the method has the advantages of reducing the production cost and reducing the number of clinical experiments. Meanwhile, compared with the expression of the recombinant antibody by a mammalian cell line, the method has the advantages of simple operation and lower production. Has remarkable economic and social benefits.

Drawings

FIG. 1: SDS-PAGE, purified fusion expressed a-1,2 mannosidase. M protein mark (Bio-Rad catalog: 161-0371); 1: 6 His-TrMannl; 2, specific antibody after glycosidase enzyme digestion.

FIG. 2: molecular schematic of specific antibodies. The tetravalent specific antibody is linked by two pairs of disulfide bonds from chains I and II, A, B and C being three Fv regions respectively.

FIG. 3: HPLC profile of specific antibodies.

Detailed Description

In order that the technical contents of the present invention can be more clearly understood, the following specific examples are specifically enumerated. However, the specific embodiments are merely illustrative, and not restrictive of the invention.

Example 1 recombinant expression and purification of glycosidase.

1.1 construction of a plasmid expressing a sequence for glycosidase

The nucleotide sequence (SEQ ID NO: 1) of Trichoderma reesei a-1,2mannosidase (Genebank: XP _006962575), which encodes 526 amino acids, was biosynthesized from Shanghai Jie Rui, then MscI cleavage site was added to the 5-and XhoI cleavage site was added to the 3-termini of the sequence, finally these sequences were loaded into pET20a vector and transformed into E.coli BL21(DE)3, and the product obtained after expression was named 6 His-TrMannl (SEQ ID NO: 1) with a molecular weight of about 67KD due to the presence of Trx and 6 His tags.

SEQ ID NO: 1Trichoderma reesei (Genebank: XP _006962575), wherein the 5-end boxed CATATG is an NdeI cleavage site, and the 3-end boxed CTCGAG is an XhoI cleavage site.

1.2 fusion protein expression and expression

Refer to Novagen pET system manual 10th, induced expression of fusion protein was performed.

Referring to Affinity Chromatography-Tagged Proteins protocol of GE, Ni Sepharose was used^TM6Fast Flow (GE, cat # 17-5318-02) purification of the expression product. The purified product was ultrafiltered using a 30KD ultrafiltration tube (millipore) at 3500rpm for 20min, the buffer was changed to 20mM PB, and the purified sample was examined by SDS-PAGE (see fig. 1).

1.3 assay of 6 His-TrMannl protein concentration with reference to nano (Thermo Co.) manipulator.

EXAMPLE 2 obtaining of fully humanized sequence

2.1 obtaining of ScFv sequence

According to previous studies by the applicant, the following optimized human sequences (sequence A, sequence B and sequence C) were obtained by optimization, wherein these sequences specifically optimized by the applicant are suitable for expression in Pichia pastoris and do not contain sequences for EcoRI, SalI, BglII and NotI restriction sites.

A sequence

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

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYASWVKGRFTISRDNAKNSLYLQXNSLRAEDTALYYCAKDIGYCTGGVCPQA

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

QFYADSAPLCVGVSGEDGNHLLHPQQWQHCQQLCAVVPTAPGQFPTTVIYEDHQRPSGIPDRFSASIDSSSNSASLIISGLKTEDEADYYCQSYDTNNRVFGGGTKLTVLG

A sequence heavy chain variable region nucleic acid sequence (A-H-N sequence, SEQ ID NO: 4)

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACTACTACATGAGCTGGATCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCGGCAGCACCATCTACTACGCCAGCTGGGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACAGCCTGTACCTGCAGAACAGCCTGAGAGCCGAGGACACCGCCCTGTACTACTGCGCCAAGGACATCGGCTACTGCACCGGCGGCGTGTGCCCCCAGGCC

A sequence light chain variable region nucleic acid sequence (A-L-N sequence, SEQ ID NO: 5)

CAGTTCTACGCCGACAGCGCCCCCCTGTGCGTGGGCGTGAGCGGCGAGGACGGCAACCACCTGCTGCACCCCCAGCAGTGGCAGCACTGCCAGCAGCTGTGCGCCGTGGTGCCCACCGCCCCCGGCCAGTTCCCCACCACCGTGATCTACGAGGACCACCAGAGACCCAGCGGCATCCCCGACAGATTCAGCGCCAGCATCGACAGCAGCAGCAACAGCGCCAGCCTGATCATCAGCGGCCTGAAGACCGAGGACGAGGCCGACTACTACTGCCAGAGCTACGACACCAACAACAGAGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGC

B sequence

B sequence heavy chain variable region protein sequence (B-H-P sequence, SEQ ID NO: 6)

EFGLSWVFLVALLRGVQCQAQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRLTISRDNSKNTLYLQMNSLRAEDTAVYYCARETSVRRDYRDYPMTDYWGQGTLVTSS

B sequence light chain variable region protein sequence (B-L-P sequence, SEQ ID NO: 7)

DMGGSKLSFFLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQRISSFLNWYQQKPGKAPKLLIYASSLQSGVPSSRFSGSGSGTDFTLTISSLQPEDVATYYCQQSYSTPPRTFGQGTKVEIKR

B sequence heavy chain variable region nucleic acid sequence (B-H-N sequence, SEQ ID NO: 8)

GAGTTCGGCCTGAGCTGGGTGTTCCTGGTGGCCCTGCTGAGAGGCGTGCAGTGCCAGGCCCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGGCATGCACTGGGTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCAGACTGACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGAGACCAGCGTGAGAAGAGACTACAGAGACTACCCCATGACCGACTACTGGGGCCAGGGCACCCTGGTGACCAGCAGC

B sequence light chain variable region nucleic acid sequence (B-L-N sequence, SEQ ID NO: 9)

GACATGGGCGGCAGCAAGCTGAGCTTCTTCCTGGGCCTGCTGCTGCTGTGGCTGAGAGGCGCCAGATGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCAGAGCCAGCCAGAGAATCAGCAGCTTCCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCCCCAGAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGAGA

C sequence

C sequence heavy chain variable region protein sequence (C-H-P sequence, SEQ ID NO: 10)

QVQLQESGPGLVKPSETLSLTCTVSGGFISDYYWNWIRQSPGKGLEWIGNIYNRGSANSSPSLKSRVTMSVDTSKNQFSLNLSSATAADTAVYYCARTRLYDSGGFSFRQFRQGFDVWGLGTVVT

C sequence light chain variable region protein sequence (C-L-P sequence, SEQ ID NO: 11)

DMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP

C sequence heavy chain variable region nucleic acid sequence (C-N-P sequence, SEQ ID NO: 12)

CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCTTCATCAGCGACTACTACTGGAACTGGATCAGACAGAGCCCCGGCAAGGGCCTGGAGTGGATCGGCAACATCTACAACAGAGGCAGCGCCAACAGCAGCCCCAGCCTGAAGAGCAGAGTGACCATGAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAACCTGAGCAGCGCCACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGAACCAGACTGTACGACAGCGGCGGCTTCAGCTTCAGACAGTTCAGACAGGGCTTCGACGTGTGGGGCCTGGGCACCGTGGTGACC

C sequence light chain variable region nucleic acid sequence (C-L-N sequence, SEQ ID NO: 13)

GACATGAGAGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGAGAGGCGCCAGATGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCAGAGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCC

2.2 Pichia expression and purification of ScFv-IgG

2.2.1 Signal peptide:

the signal peptide of the antibody is the signal peptide of the mouse kappa chain, and the polypeptide sequence is shown in sequence 14(SEQ ID NO: 14), and the corresponding nucleotide sequence is shown in sequence 15(SEQ ID NO: 15)

Leader peptide sequence of murine kappa chain amino acid sequence (SEQ ID NO: 14)

METDTLLLWVLLLWVPGSTG

Leader peptide sequence encoding the murine kappa chain nucleic acid sequence (SEQ ID NO: 15)

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT

2.2.2Fc + hinge region sequences

The amino acid sequence 221-446 (EU coding system) of human IgG1(IMGT designated IGHG1) comprises the three regions 221-234 as the Hinge region and 234-341 as the C region _H2 and 342-446 is C_H3 region (the polypeptide sequence is shown in sequence 16, namely SEQ ID NO: 16, and the corresponding nucleotide sequence is shown in sequence 17, namely SEQ ID NO: 17).

Amino acid sequence 221-446 of IGHG1 (EU coding system, SEQ ID NO: 16)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Amino acid sequence 221 of IGHG 1-a nucleotide sequence of section (EU coding system) (SEQ ID NO: 17)

GACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG

2.2.3 chaining of VH and VL (linker)

The VL and VH are linked by a linker peptide whose amino acid sequence is shown in SEQ ID NO: 18 and the corresponding nucleotide sequence is shown in SEQ ID NO: 19

VL and VH Linked peptide (SEQ ID NO: 18)

GGGGSGGGGSGGGGS

Nucleotide sequence of a linker peptide of VL and VH (SEQ ID NO: 19)

GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC

2.2.4 expression of the antibody ScFv-IgG, respectively

2.2.4.1 expression vector construction

Pairs of sequences were first paired: the method comprises the following steps: SEQ ID NO: 15+ sequence 5(a-L-N) + sequence SEQ ID NO: 19+ sequence 4 (A-H-N); and B, SEQ ID NO: 15+ sequence 9(B-L-N) + sequence SEQ ID NO: 19+ sequence 8 (B-H-N); and the third is SEQ ID NO: 15+ sequence 13(C-L-N) + sequence SEQ ID NO: 19+ sequence 12 (C-H-N); then, the sequence 17 is used to connect the above sequence pairs in order to construct the Pichia expression vector. For example, a vector expressing the fragment a antibody was constructed: SEQ ID NO: 15+ sequence 5(a-L-N) + sequence SEQ ID NO: 19+ sequence 4(a-H-N) + SEQ ID NO: 17, and the rest is analogized. Please Shanghai Czeri to synthesize these sequences separately, add EcoRI cleavage site at 5-terminal and XhoI cleavage site at 3-terminal of these sequences, and finally, insert these sequences into pPIC9k vector.

2.2.4.2 vector linearization and Positive clone screening

Referring to the invitrogen yeast operating manual, after the plasmid was linearized with SalI, the plasmid DNA was introduced by electroporation, and positive clones were selected using MD plates.

2.2.4.3 expression of antibody ScFv-IgG

A single colony is selected, firstly cultured in 10ml YPD medium at 30 ℃ and 300rpm for 24 hours, then the strain is transferred to a 1L shake flask and cultured at 30 ℃ and 300rpm until OD600 is 2-6.

B, centrifuging at 1500-3000 g for 5min at room temperature, collecting thallus, adding 1L YPM (containing methanol 1%) culture medium, and placing on a shaking table at 250-300rpm at 28-30 ℃ for induction expression.

C, adding 100% methanol into the culture medium every 24h until the final concentration is 0.5-1.0%, and inducing for 120h in total;

d, performing centrifugal separation on supernatant of the secretion expression sample, and taking the supernatant for the following purification;

2.2.4.4 purification of antibodies

All Fc domain-bearing antibodies were captured from the expression supernatant using a proteinA affinity chromatography column (shanghai borghun). After equilibration of the column with equilibration buffer (20mM PB, pH7.0), the expression supernatant was filtered through a 0.22. mu.M filter and passed through an affinity column, which was washed with equilibration buffer to an OD280 of less than 0.05 and then eluted with elution buffer (20mM glycine/hydrochloric acid, pH 3.0).

The eluted protein was subjected to SP-Sepharose cation exchange chromatography (Shanghai Bogelong Co.) to effect separation of the target bispecific antibody from the by-products. SP-after equilibration of the column with equilibration buffer A (20mM PB, pH 6.0), dilution of the sample with purified water to a conductivity between 3.0 and 3.5ms, after binding on the SP column, linear elution with elution buffer B (20mM PB,1.5M NaCL pH 6.0);

finally, the concentrate was ultrafiltered and replaced with buffer containing 0.15NaCl, 50mM sodium citrate (pH 5.5).

2.3 determination of neutralizing Activity of Single or multiple antibodies

The assay method is shown in example 4.

The measurement result shows that A, B and C respectively have neutralization protection effect, and the potency of the A, B and C respectively is 200IU/mg, 150IU/mg and 120 IU/mg. The combination has synergistic neutralization protection effect: the titer of the group A and the group B is 350 IU/mg; the titer of the group B and the group C is 280 IU/mg; the titer of the group A, the titer of the group B and the titer of the group C is 500IU/mg, wherein the group C has the best protective effect, and the titer of the group C is higher than the titer of 400 IU/mg and 450IU/mg of the prior horse tetanus F (ab)2 titer (because the molecular weight of the horse tetanus F (ab)2 titer is small) and 200IU/mg of human Tetanus Immunoglobulin (TIG). Therefore, in the next experiment, the a + B +2C trivalent antibody was further expressed.

EXAMPLE 3 acquisition of trivalent specific antibodies

3.1 expression vector construction of trivalent specific antibody

3.1.1Fc + hinge region knob mutant (or hole mutant) sequence

In the present invention, the literature (natural biotechnology (1998),16(7):677-_H3 (the polypeptide sequence is shown in SEQ ID NO: 20, and the corresponding nucleotide sequence is shown in SEQ ID NO: 21), one chain expresses a knob mutant of S354C and T366W (the polypeptide sequence is shown in SEQ ID NO: 21), and one chain expresses a hole mutant of Y349C, T366S, L368A and Y407V (the polypeptide sequence is shown in SEQ ID NO: 22, and the corresponding nucleotide sequence is shown in SEQ ID NO: 23).

Fc amino acid sequence (SEQ ID NO: 20) of sequence 20 hinge region + Knob mutant (S354C, T366W)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Sequence 21 nucleotide sequence (SEQ ID NO: 21) GACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGTGGTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG encoding the Fc of hinge region + Knob mutant (S354C, T366W)

Fc amino acid sequence (SEQ ID NO: 22) of sequence 22 hinge region + Hore mutant (Y349C, T366S, L368A and Y407V)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Sequence 23 nucleotide sequence encoding Fc for hinge region + Hore mutant (Y349C, T366S, L368A and Y407V) (SEQ ID NO: 23)

GACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTGCACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGAGCTGCGCCGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGGTGAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG

3.1.2 composition of the chains I and II of the units

Because the protection force of C is weak, when the specific antibody is constructed, a copy of C fragment is added, so that the specific antibody presents a symmetrical structure.

Composition of chain I: fragment A + Fc + hinge region knob mutant + C fragment;

the base sequence corresponding to the I chain is as follows:

a: SEQ ID NO: 15+ sequence 5(a-L-N) + sequence SEQ ID NO: 19+ sequence 4 (A-H-N);

fc + hinge region knob mutant: SEQ ID NO: 21

C fragment: sequence 12(C-H-N) + SEQ ID NO: 19+ sequence 13(C-L-N)

Composition of chain II: b fragment + Fc + hinge region or hole mutant + C fragment.

The base sequence corresponding to the II chain is as follows:

b: the amino acid sequence of SEQ ID NO: 15+ sequence 9(B-L-N) + sequence SEQ ID NO: 19+ sequence 8 (B-H-N);

fc + hinge region or hole mutant: SEQ ID NO: 23

C fragment: sequence 12(C-H-N) + SEQ ID NO: 19+ sequence 13(C-L-N)

Wherein I and II are symmetrical and can be interchanged, and the positions of A, B and C are also symmetrical and can be interchanged. The structure of the device is shown schematically (figure 2).

3.1.3 screening and purifying the positive clone of the trivalent specific antibody,

with reference to example 2.2.4.2, pPIC₉The k vector is linearized by SalI and BglII respectively, and transferred to an MD plate containing G418 simultaneously, and the Pichia pastoris positive clone which expresses I and II chains simultaneously is screened at one time.

Expression of Positive clones reference example 2.2.4.3

Purification of trivalent specific antibody reference example 2.2.4.4

The protein concentration of the bispecific antibody was determined with reference to the nano (Thermo corporation) manipulator.

3.2 glycosidase (6 × His-TrMannl) treatment of trivalent specific antibodies

The method of reference (appl microbiological Biotechnol (2015)99(9): 3913-3927) in 20mM NaAC (pH5.0) containing 0.4mM Zn2+, the ratio of antibody to glycosidase was 1: 3000 were added and digested at 37 ℃ for 16 hours.

The digested sample was purified using protein A affinity column 2.2.4.4, and the product is shown in FIG. 1.

3.3 HPLC detection

Referring to the specification of agilent 1200Infinity II and chinese pharmacopoeia 2015 edition, a column G3000SW (TOSOH corporation, japan) was used.

1. Starting HPLC, entering a workstation after the instrument is subjected to self-inspection, and selecting a method (setting the wavelength at 280nm, the column temperature at 25 ℃, the flow rate at 0.6ml/min and the running time at 40 min).

2. Taking a base line: the column was rinsed with 10% acetonitrile and replaced with mobile phase (pH 7.0, 0.2mol/L phosphate buffer containing 1% isopropanol) to equilibrate to baseline.

3. Preparation of a test solution: the sample was diluted with the mobile phase to a solution containing 12mg of protein per 1 ml.

4. Injecting 20 μ l of diluted sample solution into high performance liquid chromatograph for analysis, with running time of 40min, and determining quality by retention time, and calculating content by area normalization method.

The result shows that the purity of the purified trivalent bispecific antibody subjected to glycosidase enzyme cleavage can reach more than 85 percent by HPLC detection (see figure 3)

Example 4 measurement of antibody titer by the mouse method

The test was performed using the mouse experimental method with reference to the general rules 3508 of the chinese pharmacopoeia 2015 edition.

1. Tetanus antitoxin standard (purchased from the Chinese food and drug testing institute) preparation: diluted with borate buffered saline (pH 7.2,50mM boric acid, 0.15M sodium chloride) to give 0.5IU per ml and 0.1IU per 0.4ml of mixed solution when mixed with equal amounts of toxin.

2. Tetanus toxin standard (purchased from the Chinese food and drug testing institute) preparation: the test was diluted with borate buffered saline to give 5 test doses per ml (0.5L +, L + being the lowest lethal dose of tetanus toxin to 100% death of mice), i.e. 1 test dose per 0.4ml injection (0.1L +) after mixing with an equivalent amount of antitoxin.

The test sample to be tested is diluted with borate buffered saline to several dilutions, such that each ml contains about 0.5IU (i.e., 0.5IU) or more and less, i.e., about 0.1IU or more and less per 0.4ml injection after mixing with equal amounts of toxin. The interval between dilutions was about 5%.

3. Adding equivalent dilution test toxin into tetanus antitoxin standard and test samples to be detected with different dilutions, mixing uniformly, placing in a water bath tank, combining for 1 hour at 37 ℃, and injecting immediately.

When in injection, the injection is sequentially carried out from high dilution to low dilution, each dilution is respectively injected with 0.4ml under the abdomen or thigh root of 17-19g of mice, and each dilution is respectively injected with at least 3 mice.

4. Results observations the test mice should be observed at least once daily in the morning and afternoon for 5 consecutive days, and morbidity and mortality are recorded.

And (4) according to a result judgment method, the control mice die within 72-120 hours. The potency of the test sample to be tested should be the highest dilution at which the control mice die simultaneously or have the most severe tetanus neurotoxicity symptoms.

The experimental result shows that the titer of the expression product can reach 600IU/mg after the expression product is purified.

Sequence listing

<110> Xiamen medical college

<120> fully humanized trivalent specific antibody for neutralizing tetanus toxin and preparation method thereof

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1578

<212> DNA

<213> Trichoderma reesei(trichoderma reesei)

<400> 1

catatgagat tccctagcag ctccgtcctt gccctcgggc tcatcggacc tgcgctggcg 60

tatccaaagc cgggcgccac aaaacgtgga tctcccaacc ctacgagggc ggcagcagtc 120

aaggccgcat tccagacgtc gtggaacgct taccaccatt ttgcctttcc ccatgacgac 180

ctccacccgg tcagcaacag ctttgatgat gagagaaacg gctggggctc gtcggcaatc 240

gatggcttgg acacggctat cctcatgggg gatgccgaca ttgtgaacac gatccttcag 300

tatgtaccgc agatcaactt caccacgact gcggttgcca accaaggcat ctccgtgttc 360

gagaccaaca ttcggtacct cggtggcctg ctttctgcct atgacctgtt gcgaggtcct 420

ttcagctcct tggcgacaaa ccagaccctg gtaaacagcc ttctgaggca ggctcaaaca 480

ctggccaacg gcctcaaggt tgcgttcacc actcccagcg gtgtcccgga ccctaccgtc 540

ttcttcaacc ctactgtccg gagaagtggt gcatctagca acaacgtcgc tgaaattgga 600

agcctggtgc tcgagtggac acggttgagc gacctgacgg gaaacccgca gtatgcccag 660

cttgcgcaga agggcgagtc gtatctcctg aatccaaagg gaagcccgga ggcatggcct 720

ggcctgattg gaacgtttgt cagcacgagc aacggtacct ttcaggatag cagcggcagc 780

tggtccggcc tcatggacag cttctacgag tacctgatca agatgtacct gtacgacccg 840

gttgcgtttg cacactacaa ggatcgctgg gtccttgctg ccgactcgac cattgcgcat 900

ctcgcctctc acccgtcgac gcgcaaggac ttgacctttt tgtcttcgta caacggacag 960

tctacgtcgc caaactcagg acatttggcc agttttgccg gtggcaactt catcttggga 1020

ggcattctcc tgaacgagca aaagtacatt gactttggaa tcaagcttgc cagctcgtac 1080

tttgccacgt acaaccagac ggcttctgga atcggccccg aaggcttcgc gtgggtggac 1140

agcgtgacgg gcgccggcgg ctcgccgccc tcgtcccagt ccgggttcta ctcgtcggca 1200

ggattctggg tgacggcacc gtattacatc ctgcggccgg agacgctgga gagcttgtac 1260

tacgcatacc gcgtcacggg cgactccaag tggcaggacc tggcgtggga agcgttcagt 1320

gccattgagg acgcatgccg cgccggcagc gcgtactcgt ccatcaacga cgtgacgcag 1380

gccaacggcg ggggtgcctc tgacgatatg gagagcttct ggtttgccga ggcgctcaag 1440

tatgcgtacc tgatctttgc ggaggagtcg gatgtgcagg tgcaggccaa cggcgggaac 1500

aaatttgtct ttaacacgga ggcgcacccc tttagcatcc gttcatcatc acgacggggc 1560

ggccaccttg ctctcgag 1578

<210> 2

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Ser Trp Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Xaa Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gly Tyr Cys Thr Gly Gly Val Cys Pro Gln Ala

100 105 110

<210> 3

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Phe Tyr Ala Asp Ser Ala Pro Leu Cys Val Gly Val Ser Gly Glu

1 5 10 15

Asp Gly Asn His Leu Leu His Pro Gln Gln Trp Gln His Cys Gln Gln

20 25 30

Leu Cys Ala Val Val Pro Thr Ala Pro Gly Gln Phe Pro Thr Thr Val

35 40 45

Ile Tyr Glu Asp His Gln Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Ala Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Ile Ile Ser Gly

65 70 75 80

Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Thr

85 90 95

Asn Asn Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 4

<211> 330

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gaggtgcagc tggtggagag cggcggcggc ctggtgaagc ccggcggcag cctgagactg 60

agctgcgccg ccagcggctt caccttcagc gactactaca tgagctggat cagacaggcc 120

cccggcaagg gcctggagtg ggtgagctac atcagcagca gcggcagcac catctactac 180

gccagctggg tgaagggcag attcaccatc agcagagaca acgccaagaa cagcctgtac 240

ctgcagaaca gcctgagagc cgaggacacc gccctgtact actgcgccaa ggacatcggc 300

tactgcaccg gcggcgtgtg cccccaggcc 330

<210> 5

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cagttctacg ccgacagcgc ccccctgtgc gtgggcgtga gcggcgagga cggcaaccac 60

ctgctgcacc cccagcagtg gcagcactgc cagcagctgt gcgccgtggt gcccaccgcc 120

cccggccagt tccccaccac cgtgatctac gaggaccacc agagacccag cggcatcccc 180

gacagattca gcgccagcat cgacagcagc agcaacagcg ccagcctgat catcagcggc 240

ctgaagaccg aggacgaggc cgactactac tgccagagct acgacaccaa caacagagtg 300

ttcggcggcg gcaccaagct gaccgtgctg ggc 333

<210> 6

<211> 142

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val

1 5 10 15

Gln Cys Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

20 25 30

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

35 40 45

Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

50 55 60

Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp

65 70 75 80

Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

85 90 95

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Glu Thr Ser Val Arg Arg Asp Tyr Arg Asp Tyr Pro

115 120 125

Met Thr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Ser Ser

130 135 140

<210> 7

<211> 132

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Asp Met Gly Gly Ser Lys Leu Ser Phe Phe Leu Gly Leu Leu Leu Leu

1 5 10 15

Trp Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser

20 25 30

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

35 40 45

Ser Gln Arg Ile Ser Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ser Ser Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln

100 105 110

Gln Ser Tyr Ser Thr Pro Pro Arg Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg

130

<210> 8

<211> 426

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gagttcggcc tgagctgggt gttcctggtg gccctgctga gaggcgtgca gtgccaggcc 60

cagctggtgg agagcggcgg cggcgtggtg cagcccggca gaagcctgag actgagctgc 120

gccgccagcg gcttcacctt cagcagctac ggcatgcact gggtgagaca ggcccccggc 180

aagggcctgg agtgggtggc cgtgatctgg tacgacggca gcaacaagta ctacgccgac 240

agcgtgaagg gcagactgac catcagcaga gacaacagca agaacaccct gtacctgcag 300

atgaacagcc tgagagccga ggacaccgcc gtgtactact gcgccagaga gaccagcgtg 360

agaagagact acagagacta ccccatgacc gactactggg gccagggcac cctggtgacc 420

agcagc 426

<210> 9

<211> 396

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gacatgggcg gcagcaagct gagcttcttc ctgggcctgc tgctgctgtg gctgagaggc 60

gccagatgcg acatccagat gacccagagc cccagcagcc tgagcgccag cgtgggcgac 120

agagtgacca tcacctgcag agccagccag agaatcagca gcttcctgaa ctggtaccag 180

cagaagcccg gcaaggcccc caagctgctg atctacgcca gcagcctgca gagcggcgtg 240

cccagcagca gattcagcgg cagcggcagc ggcaccgact tcaccctgac catcagcagc 300

ctgcagcccg aggacgtggc cacctactac tgccagcaga gctacagcac cccccccaga 360

accttcggcc agggcaccaa ggtggagatc aagaga 396

<210> 10

<211> 125

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Phe Ile Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Asn Arg Gly Ser Ala Asn Ser Ser Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Asn Leu Ser Ser Ala Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Arg Leu Tyr Asp Ser Gly Gly Phe Ser Phe Arg Gln Phe Arg

100 105 110

Gln Gly Phe Asp Val Trp Gly Leu Gly Thr Val Val Thr

115 120 125

<210> 11

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu

1 5 10 15

Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln

35 40 45

Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

100 105 110

Tyr Ser Thr Pro

115

<210> 12

<211> 375

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caggtgcagc tgcaggagag cggccccggc ctggtgaagc ccagcgagac cctgagcctg 60

acctgcaccg tgagcggcgg cttcatcagc gactactact ggaactggat cagacagagc 120

cccggcaagg gcctggagtg gatcggcaac atctacaaca gaggcagcgc caacagcagc 180

cccagcctga agagcagagt gaccatgagc gtggacacca gcaagaacca gttcagcctg 240

aacctgagca gcgccaccgc cgccgacacc gccgtgtact actgcgccag aaccagactg 300

tacgacagcg gcggcttcag cttcagacag ttcagacagg gcttcgacgt gtggggcctg 360

ggcaccgtgg tgacc 375

<210> 13

<211> 348

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gacatgagag tgcccgccca gctgctgggc ctgctgctgc tgtggctgag aggcgccaga 60

tgcgacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgacagagtg 120

accatcacct gcagagccag ccagagcatc agcagctacc tgaactggta ccagcagaag 180

cccggcaagg cccccaagct gctgatctac gccgccagca gcctgcagag cggcgtgccc 240

agcagattca gcggcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctgcag 300

cccgaggact tcgccaccta ctactgccag cagagctaca gcaccccc 348

<210> 14

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 15

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

<210> 16

<211> 227

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 17

<211> 681

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg ccccagcgtg 60

ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc 120

tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 180

ggcgtggagg tgcacaacgc caagaccaag cccagggagg agcagtacaa cagcacctac 240

agggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 300

tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga agaccatcag caaggccaag 360

ggccagccca gggagcccca ggtgtacacc ctgcccccca gcagggacga gctgaccaag 420

aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 480

tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 540

gacggcagct tcttcctgta cagcaagctg accgtggaca agagcaggtg gcagcagggc 600

aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 660

ctgagcctga gccccggcaa g 681

<210> 18

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 19

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45

<210> 20

<211> 227

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 21

<211> 681

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg ccccagcgtg 60

ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc 120

tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 180

ggcgtggagg tgcacaacgc caagaccaag cccagggagg agcagtacaa cagcacctac 240

agggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 300

tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga agaccatcag caaggccaag 360

ggccagccca gggagcccca ggtgtacacc ctgcccccct gcagggacga gctgaccaag 420

aaccaggtga gcctgtggtg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 480

tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 540

gacggcagct tcttcctgta cagcaagctg accgtggaca agagcaggtg gcagcagggc 600

aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 660

ctgagcctga gccccggcaa g 681

<210> 22

<211> 227

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 23

<211> 681

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg ccccagcgtg 60

ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc 120

tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 180

ggcgtggagg tgcacaacgc caagaccaag cccagggagg agcagtacaa cagcacctac 240

agggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 300

tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga agaccatcag caaggccaag 360

ggccagccca gggagcccca ggtgtgcacc ctgcccccca gcagggacga gctgaccaag 420

aaccaggtga gcctgagctg cgccgtgaag ggcttctacc ccagcgacat cgccgtggag 480

tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 540

gacggcagct tcttcctggt gagcaagctg accgtggaca agagcaggtg gcagcagggc 600

aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 660

ctgagcctga gccccggcaa g 681

Claims

1. A fully humanized trivalent specific antibody with the function of neutralizing tetanus toxoid, characterized in that,

the trivalent specific antibody comprises a single chain I and a single chain II, wherein each single chain respectively comprises 2 single-chain variable fragments, a hinge region of 1 individual IgG1 and 1Fc fragment, and the four single-chain variable fragments respectively consist of A, B and C fragments which can respectively play a neutralizing role;

wherein, the composition of the single chain I is as follows: fragment A + Fc + hinge region knob mutant + C fragment;

the corresponding amino acid sequence of the single chain I is as follows:

the A fragment: SEQ ID NO: 14+ SEQ ID NO: 3+ SEQ ID NO: 18+ SEQ ID NO: 2;

the Fc + hinge region knob mutant: SEQ ID NO: 20;

the C fragment: the amino acid sequence of SEQ ID NO: 10+ SEQ ID NO: 18+ SEQ ID NO: 11;

composition of single chain II: b fragment + Fc + hinge region or hole mutant + C fragment;

the corresponding amino acid sequence of single strand II is:

the B fragment: the amino acid sequence of SEQ ID NO: 14+ SEQ ID NO: 7+ SEQ ID NO: 18+ SEQ ID NO: 6;

fc + hinge region or hole mutant: SEQ ID NO: 22;

c fragment: SEQ ID NO: 10+ SEQ ID NO: 18+ SEQ ID NO: 11.

2. the fully humanized trivalent specific antibody for neutralizing tetanus toxoid according to claim 1, wherein the trivalent specific antibody is expressed in the same strain of pichia pastoris and is obtained by treating a purified product with glycosidase.

3. The trivalent specific antibody according to claim 1, wherein the hinge region of (I) single chain I and single chain II are linked by two disulfide bonds; ② the human IgG Fc fragment of single chain I and the human IgG Fc of single chain II are connected by knob-into-holes.

4. A composition comprising a trivalent specific antibody according to any one of claims 1 or 2.

5. A nucleic acid molecule encoding a trivalent specific antibody according to any one of claims 1 or 2.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. Use of a trivalent specific antibody according to any one of claims 1 or 2 for the preparation of a diagnostic reagent for the detection of tetanus toxoid antigen; and/or

The application of the immune reagent as a passive immune reagent for replacing tetanus antitoxin, horse tetanus immunoglobulin or human tetanus immunoglobulin.

8. A method of producing a trivalent specific antibody according to any one of claims 1 or 2, comprising the steps of:

1) synthesizing the nucleotide sequence corresponding to the antibody sequence according to the antibody sequence,

2) transferring the sequence synthesized in the step 1) into pichia pastoris by adopting a carrier, and selecting a pichia pastoris positive clone;

3) expressing trivalent specific antibody in Pichia pastoris;

4) treating the trivalent specific antibody by using a recombinantly expressed glycosidaseTrichoderma reeseiA-1,2mannosidase of (1);

wherein, the nucleotide sequence in the step 1) comprises:

composition of single-chain I strand: fragment A + Fc + hinge region knob mutant + C fragment;

the base sequence corresponding to the I chain comprises:

A：SEQ ID NO：15+ SEQ ID NO：5+ SEQ ID NO：SEQ ID NO：19+ SEQ ID NO：4；

fc + hinge region knob mutant: SEQ ID NO: 21

C fragment: the amino acid sequence of SEQ ID NO: 12+ SEQ ID NO: 19+ SEQ ID NO: 13;

composition of chain II: b fragment + Fc + hinge region or hole mutant + C fragment;

the base sequence corresponding to the II chain is as follows:

B：SEQ ID NO：15+ SEQ ID NO：9+ SEQ ID NO：19+ SEQ ID NO：8；

fc + hinge region or hole mutant: SEQ ID NO: 23

C fragment: SEQ ID NO: 12+ SEQ ID NO: 19+ SEQ ID NO: 13;

in the step 1), the nucleotide sequences of the single chain I and the single chain II are respectively synthesized, and in the step 3), pichia pastoris positive clones which simultaneously express the single chain I and the single chain II are screened out.