KR100263438B1 - FAB Region Genes and Sequences of Mouse Aggregated Monoclonal Antibodies Against Human Blood Cells - Google Patents

Abstract

PURPOSE: A gene encoding fab region of rat's monoclonal antibody against human hemocyte is provided which is useful in diagnosing antigens in blood of HIV, HBV, CMV, and other virus when expressed with human genes of antibodies. CONSTITUTION: The gene encoding the light chain of fab region of rat's monoclonal antibody against human hemocyte is composed of CDR1(Lys, Ala, Ser, Gln, lle, Val, Gly, Thr, Asn, Val, Ala), CDR2(Ser, Ala, Ser, Tyr, Arg, Tyr, Ser), and CDR3(Gln, Gln, Tyr, Asn, Arg, Tyr, Pro, Trp) and its cDNA is represented by the sequence ID No. 3 of 5'-GAGGTCCAGA TGCAACAGTC TGGA-3'. The gene encoding the heavy chain of the fab region is composed of CDR1(Ser, Tyr, Tyr, Met, Tyr), CDR2(Tyr, Ile, Ser, Cys, Tyr, Asn, Gly, Ala, Thr, Thr, Tyr, Asn, Arg, Lys, Phe, Lys, Gly) and CDR3(HIs, Tyr, Tyr, Gly, Ser, Pro, Leu, Asp, Tyr) and its cDNA is represented by sequence ID. No. 4 of 5'-AATTTTCTTG TCCACCTTGG TGCT-3'. Plasmid vectors, pYBM-AL and pYBM-AH, containing cDNAs transforms E.coli to obtain transformants, DH5α/pYBM-AL(KCTC 8857P) and DH5α/pYBM-AH (KCTC 8856P).

Description

FaF site gene and its nucleotide sequence of mouse aggregated monoclonal antibody against human blood cells

The present invention relates to Fab sites of mouse aggregated monoclonal antibodies against human blood cells, cDNA genes encoding them and their nucleotide sequences. Specifically, the present invention relates to Fab sites of mouse aggregated monoclonal antibodies against human blood cells, heavy and light chain cDNA genes encoding them, their base sequences, and plasmid vectors comprising the same, wherein the genes are in blood such as HIV. And humanized antibodies for diagnosing antigen.

In the diagnosis and treatment of human diseases, immunological methods using antigen-antibody reactions are widely used. Antibodies to specific antigens used in antigen-antibody reactions, etc. can be classified into the following two categories. First, an antibody generated by an immune response that specifically recognizes only one epitope present on the antigen surface is monoclonal. An antibody is called an antibody, and an antibody produced by an immune response that recognizes one or more epitopes is called a polyclonal antibody. Since monoclonal antibodies specifically react only to specific epitopes, the response is evident in disease diagnosis and immunological treatment.

Monoclonal antibodies are obtained by preparing spleen cells and hematopoietic cancer cells (myeloma) that produce antibodies that specifically bind to antigens such as viruses, and making and culturing hybridoma cells.

In recent years, attempts have been made to improve monoclonal antibodies into chimeric antibodies, such as complex antibodies in which heterologous antigens are chemically bound to antibodies, or regulated genes at sites that bind antigens. The proportion is increasing (Winter & Milatien, Nature , 349, 293, 1991; Better et al., Science , 240, 1041, 1988).

In fact, attempts have been made to improve monoclonal antibodies for diagnosing infections of various viruses. For example, monoclonal antibodies for diagnosing HIV-1 infection are chemically combined with HIV-1 antigen and hemagglutinating antibodies. Have been reported (Kemp et al., Science, 244, 1352, 1988).

In order to diagnose an infection of human virus, monoclonal antibodies made from animal cells such as mice have an adverse effect that induces an immune response when injected into humans. Humanized antibodies should be made. Humanized antibodies can be prepared by implanting a region of complementarity determining regions (CDRs) that directly bind to an antigen in a variable region of a mouse monoclonal antibody. To this end, a mouse antibody variable region is obtained and expressed in animal cells by fusion with genes of human antibodies by genetic recombination.

In order to improve diagnostic methods for viral infections, we have developed nine mouse clones from mice that produce monoclonal antibodies against human erythrocytes, and hemagglutination among five clones producing IgG. A relatively fast clone, # 12, was selected and named HAG 12 (Nham et al., Mol. Cells , 3, 221, 1993).

In addition, the present inventors continued to develop a method for effectively preventing and diagnosing a viral infection, and as a result, cloned the genes of the mouse aggregate antibody Fab region against human blood cells useful for developing humanized antibodies and the like in the mouse cell clone HAG 12. The present invention was completed by determining its nucleotide sequence and confirming its properties.

It is an object of the present invention to provide a Fab region of a mouse aggregated monoclonal antibody against human blood cells, a cDNA gene encoding the same, and a nucleotide sequence thereof.

Figure 1 shows the structure of the vector pYBM-AL containing the Fab site light chain cDNA 645 base pair of the mouse aggregate monoclonal antibody against human blood cells,

Figure 2 shows the structure of the vector pYBM-AH containing the Fab site heavy chain cDNA 645 base pair of the mouse aggregated monoclonal antibody against human blood cells.

The present invention provides CDR1 (Lys, Ala, Ser, Gln, Ile, Val, Gly, Thr, Asn, Val, Ala), CDR2 (Ser, Ala, Ser, Tyr, Arg, Tyr, Ser) and CDR3 (Gln, Gln , Tyr, Asn, Arg, Tyr, Pro, Trp) provides a gene encoding the light chain of the Fab region of the mouse aggregated monoclonal antibody against human blood cells. Specifically, the present invention provides a light chain cDNA gene encoding the Fab region of a mouse aggregated monoclonal antibody against human blood cells and the nucleotide sequence of SEQ ID NO: 5.

In addition, the present invention provides CDR1 (Ser, Tyr, Tyr, Met, Tyr), CDR2 (Tyr, Ile, Ser, Cys, Tyr, Asn, Gly, Ala, Thr, Thr, Tyr, Asn, Arg, Lys, Phe, Lys, Gly) and CDR3 (His, Tyr, Tyr, Gly, Ser, Pro, Leu, Asp, Tyr) provide genes encoding the heavy chain of the Fab region of the mouse aggregated monoclonal antibody against human blood cells. Specifically, the present invention provides a heavy chain cDNA gene encoding the Fab region of a mouse aggregated monoclonal antibody against human blood cells and the nucleotide sequence of SEQ ID NO: 6.

In addition, the present invention provides a plasmid vector pYBM-AL comprising a light chain gene of the monoclonal antibody and an E. coli transformant thereof (Accession Number: KCTC 8857P) and a plasmid vector pYBM-AH comprising a heavy chain gene and an E. coli transformant thereof. (Accession No .: KCTC 8856P).

The present invention also provides an amino acid sequence of a mouse aggregate monoclonal antibody consisting of a light chain of SEQ ID NO: 7 and a heavy chain of SEQ ID NO: 8 binding to human blood cells.

Hereinafter, the present invention will be described in detail.

The present invention uses a mouse cell clone HAG 12 which produces a Fab region of a monoclonal antibody to obtain a gene encoding the Fab region of a mouse aggregated monoclonal antibody against human blood cells.

Specifically, RNA was extracted from the mouse cell clones to synthesize cDNAs of light and heavy chain genes, and as a template, primers of SEQ ID NO: 1 and primers of SEQ ID NO: 2 for light chains, and primers of SEQ ID NO: 3 for heavy chains and SEQ ID NO: 4 Polymerase chain reaction (PCR) was performed using the primers of. As a result, the light chain cDNA gene of size 645 bp of SEQ ID NO: 5 and the heavy chain cDNA gene of size 645 bp of SEQ ID NO: 6 were isolated.

Looking at the characteristics of the light chain gene encoding the Fab region of the mouse aggregation monoclonal antibody to human blood cells of the present invention. The variable region consists of 107 amino acids, CDR1 amino acids 24-34 (Lys, Ala, Ser, Gln, Ile, Val, Gly, Thr, Asn, Val, Ala), CDR2 amino acids 50-56 (Ser, Ala, Ser, Tyr, Arg, Tyr, Ser) and CDR3 consist of amino acids 89-96 (Gln, Gln, Tyr, Asn, Arg, Tyr, Pro, Trp). The cysteine residues of the monoclonal antibody Fab region are 5, located at 23, 88, 134, 194 and 214, and 23 and 88, 134 and 194 bind to each other, and 214 binds to the heavy chain. The J element consists of amino acids 96-107 with amino acids 11 methionine, 18 arginine, 20 serine and 46 alanine. The antibody belongs to subgroup I.

Looking at the characteristics of the heavy chain gene encoding the Fab region of the mouse aggregation monoclonal antibody to human blood cells of the present invention. The variable region consists of 118 amino acids, CDR1 amino acids 31-35 (Ser, Tyr, Tyr, Met, Tyr), CDR2 amino acids 50-66 (Tyr, Ile, Ser, Cys, Tyr, Asn, Gly, Thr, Thr, Tyr, Asn, Arg, Lys, Phe, Lys, Gly) and CDR3 consist of amino acids 99-107 (His, Tyr, Tyr, Gly, Ser, Pro, Leu, Asp, Tyr). Cysteine residues are located at 22, 55, 96, 145, 200 and 133, 22 and 96, 145 and 200 bind to each other, 133 binds to the light chain. But number 55 has yet to know its role. The J element consists of amino acids 106-118 and the subgroup belongs to IIa. The other amino acid 14 is threonine.

The cDNAs encoding the heavy and light chains of the antigen-binding fragment regions of the mouse aggregated monoclonal antibody genes against human blood cells have been found to possess base sequences corresponding to the general characteristics of the antibody and its isotype. This suggests that these antibody cDNAs are functional.

The cDNA genes were cloned into plasmid vectors, respectively, to prepare plasmid vectors pYBM-AL and pYBM-AH of the present invention.

The plasmid vectors were transformed into E. coli DH5α strains and the transformants were deposited in the GenBank of the Biotechnology Research Institute affiliated with the Korea Institute of Science and Technology on November 28, 1997 (accession number: KCTC 8857P and KCTC 8856P).

The present invention provides an amino acid sequence of the mouse aggregate monoclonal antibody Fab region consisting of a light chain of SEQ ID NO: 7 and a heavy chain of SEQ ID NO: 8 that binds human blood cells.

CDRs region that directly binds to the antigen in the Fab region of the mouse monoclonal antibody according to a known method using the cDNA of the antigen-binding fragment of the mouse monoclonal antibody which specifically binds to human blood cells of the present invention and causes an aggregation reaction The humanized antibody can be prepared by implanting a human antibody into human antibodies to minimize the immune response when injected into the human body, and to produce a large amount of recombinant monoclonal antibody that binds to human red blood cells and causes aggregation. It can be used to diagnose antigens in the blood, such as HIV, HBV, CMV, and other viruses.

Hereinafter, the present invention will be described in detail by examples.

However, the embodiments are only illustrative of the present invention and the present invention is not limited by the embodiments.

<Example 1> Extraction of total RNA from mouse cell clone HAG 12

According to the Nonidet P-40 method (Favaloro et al., 1980), clone HAG12 incubated in IMDM medium with 10% calf serum was washed with phosphate buffer and RNA extract (0.14 M NaCl). , 15 mM MgCl ₂ , 0.5% NP-40, 1 mM dithiothreitol, 20 mM vanadyl ribonucleotide complex in 10 mM Tris-Cl, pH 8.6) dissolved in 10 mM Tris Suspended by adding 10 times and centrifuged. Add a volume of digestion buffer (0.3 M NaCl, 2% SDS, 25 mM EDTA in 0.2 M Tris-Cl, pH 8.0) to the supernatant to a final concentration of 200 μg / ml. Mix and treat at 37 ° C. for 1 hour. RNA was extracted twice with the same volume of phenol and precipitated by addition of ethanol to obtain total RNA.

<Example 2> Cloning of the mouse aggregated antibody Fab light chain gene to human blood cells

Referring to the N-terminal amino acid sequence of the light chain Fab of the mouse aggregated monoclonal antibody against human blood cells (Nham et al., Mol. Cells , 3, 221, 1993), primers Vk1 and SEQ ID NO: 2 having the nucleotide sequence of SEQ ID NO: 1 Primer Ck1 having a nucleotide sequence was synthesized.

1 μg of RNA prepared in Example 1 and 1 unit / ml RNA decomposing enzyme inhibitor (RNase inhibitor) were added to the buffer (50 mM KCl, 5 mM MgCl ₂ in 10 mM Tris-Cl, pH 8.3), followed by AMV reverse. 0.25 unit / ml of AMV reverse transcriptase was mixed and 100 μl of mineral oil was dropped. Using a Robocycler PCR Machine (Stratajin Co., Ltd.), the reaction was performed once at 42 ° C. 30 minutes, 99 ° C. 5 minutes, and 5 ° C. 5 minute intervals, and the buffer, 0.2 μM primer Vk1 and primer Ck1 were subjected to tack polymer. 0.5 μl of Raise (Taq polymerase (Takarasa; 2.5 unit / 100 μl)) and sterile distilled water to make a final volume of 100 μl, reacted 30 times at 94 ° C. 30 sec, 55 ° C. 30 sec, 72 ° C. 1.5 min, and then agarose gel. Electrophoresis.

Plasmid pYBM-AL was prepared by inserting the light chain unit cDNA prepared above into the restriction enzyme SmaI position of plasmid pUC19 (see FIG. 1 ).

Example 3 Cloning of Mouse Aggregated Monoclonal Antibody Fab Light Chain Gene to Human Blood Cells

The primer Vh1 having the nucleotide sequence of SEQ ID NO: 3 and the primer Ch1 having the nucleotide sequence of SEQ ID NO: 4 were synthesized by referring to the N-terminal amino acid sequence of the heavy chain Fab of the mouse aggregate monoclonal antibody against human blood cells. In the same manner as in Example 2, 1 μg of RNA prepared in Example 1 and 1 unit / ml RNA degrading enzyme inhibitor (RNase Inhibitor) were added to the buffer (50 mM KCl, 5 mM MgCl ₂ in 10 mM Tris-Cl, pH 8.3). ), 0.25 unit / ml of AMV reverse transcriptase was mixed, and 100 μl of mineral oil was dropped. React once with a Robocycler PCR machine at 42 ° C. 30 min, 99 ° C. 5 min, 5 ° C. 5 min intervals, buffer, 0.2 μM primer Vh1 and primer Ch1, Tag polymerase (Takarasa; 2.5 unit / 100 μl) ) 0.5μl and sterile distilled water to make a final volume of 100μl, 30 ℃ 94 ℃ 30 seconds, 30 ℃, 30 ℃ 72 reactions 1.5 minutes, and electrophoresed with agarose gel. The prepared heavy chain unit cDNA was inserted into the SmaI position of the plasmid pUC19 to prepare pYBM-AH (see FIG. 2 ).

<Example 4> Determination of the base sequence of the mouse aggregate monoclonal antibody gene to human blood cells

M13 consensus primers were prepared by chain termination using the deideoxy nucleotide reagent of the light chain unit and the heavy chain unit DNA of the mouse aggregate monoclonal antibody Fab against human blood cells prepared in Examples 2 and 3. Base sequences were determined (Sanger et al., Proc. Natl. Acad. Sci ., 74, 5463, 1977). The base sequence of the light chain gene is shown in SEQ ID NO: 3, and the base sequence of the heavy chain gene is shown in SEQ ID NO: 4. Upon receipt of the genes into the International GenBank / EMBL Data Library, all of the CDR1, CDR2, CDR3 and V-region DNA sequences are new nucleotide sequences that have not been identified so far. ) Was given a unique number (expected date of publication: Apr. 1998).

As described above, the cDNA genes encoding the Fab region heavy and light chains of the mouse aggregate monoclonal antibody that binds to human blood cells are functional, and when expressed in combination with human antibody genes, it is expressed in blood such as HIV, HBV, CMV, and other viruses. It can be usefully used to diagnose antigens.

(Sequence list)

SEQ ID NO: 1

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 2

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 3

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 4

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 5

Sequence length: 645

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: gene

(Sequence list)

SEQ ID NO: 6

Sequence length: 645

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: gene

(Sequence list)

SEQ ID NO: 7

Length of sequence: 214

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

(Sequence list)

SEQ ID NO: 8

Length of sequence: 215

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

Claims (10)

CDR1 (Lys, Ala, Ser, Gln, Ile, Val, Gly, Thr, Asn, Val, Ala), CDR2 (Ser, Ala, Ser, Tyr, Arg, Tyr, Ser) and CDR3 (Gln, Gln, Tyr, Gene encoding the light chain of the mouse aggregate monoclonal antibody Fab site for human blood cells consisting of Asn, Arg, Tyr, Pro, Trp). The cDNA gene according to claim 1, wherein the gene has a nucleotide sequence of SEQ ID NO: 3. CDR1 (Ser, Tyr, Tyr, Met, Tyr), CDR2 (Tyr, Ile, Ser, Cys, Tyr, Asn, Gly, Ala, Thr, Thr, Tyr, Asn, Arg, Lys, Phe, Lys, Gly) and A gene encoding the heavy chain of the mouse aggregated monoclonal antibody Fab region against human blood cells consisting of CDR3 (His, Tyr, Tyr, Gly, Ser, Pro, Leu, Asp, Tyr). The cDNA gene of claim 3, wherein the gene has a nucleotide sequence of SEQ ID NO: 4 A plasmid vector pYBM-AL comprising the cDNA gene of claim 2. A plasmid vector pYBM-AH comprising the cDNA gene of claim 4. Escherichia coli transformant transformed with the plasmid pYBM-AL of claim 5 DH5α / pYBM-AL (Accession Number: KCTC 8857P). E. coli transformant DH5α / pYBM-AH (Accession No .: KCTC 8856P) transformed with the plasmid pYBM-AH of claim 6. A mouse aggregated monoclonal antibody consisting of the light chain of SEQ ID NO: 7 and the heavy chain of SEQ ID NO: 8 binding to human blood cells. Use of the gene encoding the mouse aggregated monoclonal antibody Fab site for human blood cells of claims 1 to 4 with a human antibody gene for use in diagnosis of infection such as HIV-1.

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