CN117384284A - Recombinant monoclonal antibody and application thereof - Google Patents
Recombinant monoclonal antibody and application thereof Download PDFInfo
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- CN117384284A CN117384284A CN202311341312.4A CN202311341312A CN117384284A CN 117384284 A CN117384284 A CN 117384284A CN 202311341312 A CN202311341312 A CN 202311341312A CN 117384284 A CN117384284 A CN 117384284A
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- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 claims abstract description 18
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims abstract description 17
- 229940034208 thyroxine Drugs 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 15
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- MGJYOHMBGJPESL-UHFFFAOYSA-L disodium;1-[8-(2,5-dioxo-3-sulfonatopyrrolidin-1-yl)oxy-8-oxooctanoyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].[Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)C(S([O-])(=O)=O)CC1=O MGJYOHMBGJPESL-UHFFFAOYSA-L 0.000 description 2
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
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- XUIIKFGFIJCVMT-LBPRGKRZSA-N L-thyroxine Chemical compound IC1=CC(C[C@H]([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-LBPRGKRZSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Endocrinology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of antibody engineering, in particular to a recombinant monoclonal antibody and application thereof. The invention provides thyroxine T4 recombinant monoclonal antibody, a preparation method and application thereof. The light chain variable region of the antibody has an amino acid sequence shown as SEQ ID NO. 1, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 2. The invention discloses a method for screening and obtaining positive clones by using phage display technology. The invention discloses application of a fragmented antibody to clinical diagnosis and also discloses a preparation method of the fragmented antibody. The thyroxine T4 recombinant antibody provided by the invention has high consistency with the detection results of mainstream factories in the current market in detecting the FT4 content in serum, and plays an important role in later application and clinical diagnosis.
Description
Technical Field
The invention relates to the technical field of antibody engineering, in particular to a recombinant monoclonal antibody and application thereof.
Background
Thyroxine is synthesized and secreted by thyroid follicular epithelial cells, has bioactivity, and can promote metabolism of glycoprotein fat to produce energy and heat, and promote growth and development. Thyroxine is an important indicator of determining thyroid function and hypothalamic pituitary thyroid axis function. Thyroxine is a manifestation of hyperthyroidism, whereas it is a manifestation of hypothyroidism. Measuring thyroxine concentration in serum is one of the important means to assist in judging thyroid function and diagnosing various thyroid diseases.
T4, thyroxine, also known as tetraiodothyronine, is hydrolyzed and enters the blood, 99.98% of T4 is non-covalently bound to plasma proteins, the remainder being FT4 0.02%. While FT4 is a hormone substance that actually enters the target cell and binds to the receptor to act. The state of thyroid mechanisms is closely related to the levels of FT3 and FT4 in the circulation. Can be used for distinguishing the subclinical states of hyperthyroidism, hyponychium and first-class work. FT4 in normal human serum: 12.00 to 22.00pmol/L.
Phage display technology is not only a high-efficiency screening system, but also an in vitro maturation process, and monoclonal recombinant antibodies with improved affinity and specificity are possible to obtain through antibody gene library technology and phage display technology. Therefore, the preparation method for providing the T4 recombinant monoclonal antibody with higher affinity and specificity has important significance.
Disclosure of Invention
In view of the above, the recombinant monoclonal antibody and the application thereof provided by the invention have higher affinity, specificity and stability.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides an antibody comprising:
the light chain variable region thereof has:
(1) An amino acid sequence shown as SEQ ID NO. 1; or (b)
(2) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (1), and having the same or similar functions as those of (1); or (b)
(3) An amino acid sequence having at least 70% homology with the amino acid sequence as set forth in (1) or (2);
the heavy chain variable region thereof has:
(4) An amino acid sequence shown as SEQ ID NO. 2; or (b)
(5) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (4), and having the same or similar functions as those of (4); or (b)
(6) An amino acid sequence having at least 70% homology with the amino acid sequence as shown in (4) or (5);
the plurality is 2 to 30.
In some embodiments of the invention, the antibodies comprise single chain antibodies;
the light chain variable region of the single chain antibody has:
(i) An amino acid sequence shown as SEQ ID NO. 1; or (b)
(ii) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (i), and having the same or similar functions as (i); or (b)
(iii) An amino acid sequence having at least 70% homology with an amino acid sequence as set forth in (i) or (ii);
the heavy chain variable region of the single chain antibody has:
(iv) An amino acid sequence shown as SEQ ID NO. 2; or (b)
(v) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (iv), and having the same or similar functions as (iv); or (b)
(iv) An amino acid sequence having at least 70% homology with the amino acid sequence as set forth in (iv) or (v);
the plurality is 2 to 30.
In some embodiments of the invention, the antibody is a fragmented antibody;
the preparation method of the fragmented antibodies comprises the step of fragmenting the antibodies by using protease.
In some embodiments of the invention, the protease comprises one or more of pepsin, papain or Ides enzyme in the preparation of the fragmented antibodies in the antibodies described above.
In some embodiments of the invention, the immunogen of the above antibody is thyroxine conjugated to a carrier protein.
In some embodiments of the invention, the carrier protein in the thyroxine of the conjugated carrier protein in the above antibody comprises one or more of KLH, BSA or OVA.
The invention also provides nucleic acid molecules encoding the antibodies described above.
In some embodiments of the invention, the nucleic acid molecules described above comprise:
the device comprises:
(7) A nucleotide sequence shown as SEQ ID NO. 3; or (b)
(8) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (7), and having the same or similar function as (7); or (b)
(9) A nucleotide sequence having at least 70% homology with the nucleotide sequence as set forth in (7) or (8);
and/or
The device comprises:
(10) A nucleotide sequence shown as SEQ ID NO. 4; or (b)
(11) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (10), and having the same or similar function as (10); or (b)
(12) A nucleotide sequence having at least 70% homology with the nucleotide sequence as set forth in (10) or (11);
the plurality is 2 to 90.
The invention also provides expression vectors comprising the above nucleic acid molecules, as well as acceptable genetic elements.
The invention also provides a host cell comprising the above nucleic acid molecule or the above expression vector.
In some embodiments of the invention, the host cell comprises 293 cells and/or CHO cells.
The invention also provides a preparation method of the antibody, which comprises the following steps:
(A) Connecting the nucleic acid molecules to a framework to obtain an expression vector, introducing the expression vector into a host cell, and culturing to obtain the antibody; or (b)
(B) Introducing the expression vector into a host cell, and culturing to obtain the antibody; or (b)
(C) Culturing the host cell to obtain the antibody.
The invention also provides application of the antibody in preparation of a reagent or a kit for detecting thyroxine.
The invention also provides a reagent comprising the antibody and acceptable auxiliary materials or auxiliary agents.
The invention also provides a kit comprising the antibody or the reagent and acceptable auxiliary materials or auxiliary agents.
The invention also provides a device comprising a coating of the antibody described above, and an acceptable component.
The recombinant monoclonal antibody has the following effects:
1) Experimental results of clinical relevance show that the clinical examination lineCorrelation of samples in the sexual range, kit for self-producing fragmented antibody combination and marketed rogowski kit R 2 ＞0.98；
2) The experimental result of the stability evaluation shows that the amplitude of the signal value and the concentration value is within 10%, and the stability is good;
3) Specificity experiments show that the crossing rate of the self-produced goat monoclonal antibody and rT3 is reduced by 8 times compared with that of the commercial goat monoclonal antibody; the crossing rate of tetraiodothyroacetic acid is reduced by 5.3 times compared with that of the commercial sheep monoclonal antibody; the crossing rate of 3,5' -2 iodine-thyroxine is reduced by 41 times compared with that of the commercial sheep monoclonal antibody. The segmented antibody provided by the invention has better specificity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows agarose gel electrophoresis of extracted spleen RNA; wherein SP represents spleen; BM stands for bone marrow; marker is TAKARA DL2000, cat# 3427A;
FIG. 2 shows an agarose gel electrophoresis pattern of the VL/VH gene PCR products; wherein, SP represents spleen, and lanes of SP are marker, light chain and heavy chain in sequence from left to right; BM represents bone marrow, lanes of BM are marker, light chain and heavy chain in sequence from left to right; marker is TAKARA DL2000, cat# 3427A;
FIG. 3 shows agarose gel electrophoresis of scFv gene PCR products; wherein, lane 1 and lane 2 are multiple wells; marker is TAKARA DL2000, cat# 3427A;
FIG. 4 shows SDS-PAGE patterns of recombinant antibody purification; wherein, two lanes are multiple wells;
FIG. 5 shows an electrophoretogram of an enzyme-cleaved antibody; wherein the lanes are full-length antibody, F (ab') 2 antibody and marker in order from left to right;
FIG. 6 shows the clinical relevance of the kit of the invention.
Detailed Description
The invention discloses a recombinant monoclonal antibody and application thereof, and a person skilled in the art can refer to the content of the recombinant monoclonal antibody and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention relates to the technical field of antibody engineering, in particular to a preparation method of a thyroxine T4 antibody. The recombinant antibody prepared by the genetic engineering technology and the phage display technology can be applied to immunological detection.
One of the technical problems to be solved by the invention is to provide thyroxine T4 recombinant monoclonal antibody.
The second technical problem to be solved by the present invention is to provide a DNA molecule encoding the thyroxine T4 recombinant mab.
The third technical problem to be solved by the invention is to provide a preparation method of the monoclonal antibody.
The fourth technical problem to be solved by the present invention is to provide a method for preparing fragmented antibodies.
The fifth technical problem to be solved by the present invention is to provide the use of the monoclonal antibody.
In order to achieve the above object, the present invention provides a method for preparing thyroxine T4 recombinant monoclonal antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region has an amino acid sequence shown as SEQ ID NO. l, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 2.
In another aspect, the invention provides a DNA molecule encoding the monoclonal antibody described above.
In a preferred embodiment, the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO. 3 encoding the light chain variable region of said monoclonal antibody and the nucleotide sequence shown in SEQ ID NO. 4 encoding the heavy chain variable region of said monoclonal antibody.
In a third aspect the present invention provides an expression vector comprising a DNA sequence as described above and an expression control sequence operably linked to the sequence.
In a fourth aspect the invention provides a host cell transformed with an expression vector as described above. In a preferred embodiment, the host cell is a mammalian 293 cell.
In a fifth aspect, the invention provides a method of preparing a F (ab) 2' antibody by cleavage.
The sixth aspect of the invention provides a kit comprising thyroxine T4 recombinant monoclonal antibody.
The recombinant antibody disclosed by the invention has the advantages that the concentration of FT4 in serum of a patient can be rapidly and sensitively detected, and the recombinant antibody has important application value for clinical detection.
The specificity of the T4 goat monoclonal antibody is better than that of the goat monoclonal antibody sold in the market, and the goat monoclonal antibody can be applied to a kit.
The sequence information of the thyroxine T4 recombinant monoclonal antibody is as follows:
light chain variable region amino acid sequence (SEQ ID NO: l):
QAVLTQPSSVSGSLGQRVSITCSGSSSNIGGRLGVGWYQQVPGSGLKTIIYDTSIRSSGVPD RFSGSRSGNTATLTISSLQAEDEADYYCAALDTSTWDFLFGSGTRLTVL
heavy chain variable region amino acid sequence (SEQ ID NO: 2):
QVRLQESGPSLVKPSQTLSLTCTVSGVSLTSTAVGWVRQAPGKVPEWLGGITSGGSAVYKPA LKSRLSITRDTSMSQVSLSLSSVTTEDAAMYRCYSHWPMDSGANIGYWGPGLPVTVSL
nucleotide sequence encoding a light chain variable region (SEQ ID NO: 3):
CAGGCTGTGCTGACTCAGCCGTCCTCCGTGTCCGGGTCCCTGGGCCAGAGGGTCTCCATCACCTGCTCTGGAAGCAGCAGCAACATCGGGGGTCGTCTTGGTGTGGGCTGGTACCAACAGGTCCCAGGATCAGGCCTCAAAACCATCATCTATGATACTAGTATTCGATCCTCGGGGGTCCCGGACCGATTCTCTGGCTCCAGGTCTGGCAACACGGCCACCCTGACCATCAGCTCGCTCCAGGCTGAGGACGAGGCCGATTATTACTGTGCAGCTCTTGACACCAGTACTTGGGATTTTCTTTTCGGCAGCGGGACCAGGCTGACCGTCCTG
nucleotide sequence encoding a heavy chain variable region (SEQ ID NO: 4):
CAGGTGCGGCTGCAGGAGTCGGGACCCAGCCTGGTGAAGCCCTCACAGACCCTCTCCCTCACCTGCACGGTCTCTGGAGTCTCATTGACCAGCACTGCTGTAGGTTGGGTCCGACAGGCTCCAGGAAAGGTGCCGGAGTGGCTTGGCGGTATAACCAGCGGTGGAAGTGCAGTCTATAAACCGGCCCTGAAGTCCCGGCTAAGCATCACCAGGGACACCTCCATGAGCCAAGTCTCCCTGTCACTGAGCAGCGTGACAACTGAGGACGCGGCCATGTACAGGTGTTATAGTCATTGGCCTATGGATAGTGGTGCCAATATCGGCTACTGGGGCCCGGGACTCCCGGTCACCGTGTCCTTG
unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all commercially available and commercially available.
The invention is further illustrated by the following examples:
example 1
T4 was dissolved in 5mg/mL of ultrapure water, BS3 (bis (sulfosuccinimidyl) suberate) was dissolved in 20mg/mL of ultrapure water, KLH was dissolved in 10mg/mL of PBS 0.01mol/L, nT4: nBS3: nKLH=1000:1000:1. And (3) taking the dissolved T4 antigen, slowly dripping the dissolved T4 antigen into the KLH solution, and vibrating while adding the dissolved T4 antigen to ensure full and uniform mixing. And (3) slowly dripping the BS3 into the mixed solution, and vibrating while adding to ensure full mixing, and vibrating at room temperature after mixing for reaction overnight. (room temperature requirement, 18-27 ℃ C., reaction time not less than 16 h) and pH 7.2 (single dialysis amount 2L) with 0.01mol/L PBS, and dialyzing for 5 times for later use.
Three female sheep of about 6 months of age were immunized 4 times with the prepared antigen T4-BS 3-KLH. The immunization period is 30 days, subcutaneous multipoint injection is carried out, two sides of the back of an immunization part, the front and rear groins are treated, 4mg of immune antigen is fully emulsified with an equal volume of Freund's complete adjuvant for the first time, and 2mg of immune antigen is fully emulsified with an equal volume of Freund's incomplete adjuvant for the last three times for immunization. Blood is taken from the ear margin vein on the 10 th day after the third immunization, standing is carried out for 1h at 37 ℃, centrifugation is carried out for 10min at 6000r/min, and the supernatant (antiserum) is collected for ELISA detection of the immune effect.
Antisera titer detection: a detection plate (Costar) was prepared, and T4-BS3-BSA antigen was added to 0.05mol/L CB (pH 9.6) coating buffer at a coating concentration of 1/2k. The test serum is diluted from 1:500 in a doubling ratio, 50 mu L/hole is provided, meanwhile, the non-immunized sheep serum is used as a negative control, and the test serum is incubated for 30min at 37 ℃; PBST is washed 5 times, patted dry, added with sheep secondary antibody 1/1k,50 mu L/hole, incubated for 30min at 37 ℃; PBST was washed 5 times, dried by shaking, and added with a luminescent substrate A, B solution (50. Mu.L/well each) and reacted in the dark for 5 minutes to determine a signal value.
The potency reaches 10 5 The final booster immunization is carried out, the animals are sacrificed three days later, spleen cells are extracted, total RNA of spleen tissues is routinely extracted by a Trizol method, and cDNA is synthesized by reverse transcription.
Example 2: scFv gene splice
The light chain variable region and the heavy chain variable region of the antibody were amplified by PCR using the cDNA obtained in example 1 as a template, and the primers were as follows:
light chain primer F (SEQ ID NO: 5):
CTGGGTGCCCGGCAGCACTGGCGCC CAGGCTGTGCTGACTCAGCC
light chain primer R (SEQ ID NO: 6):
GGAGCGCTCTTAGGCTGGCC CAGGACGGTCAGCCTGGTCC
heavy chain primer F (SEQ ID NO: 7):
CTGGGTGCCCGGCAGCACTGGCGCC CAGGTGCGGCTGCAGGAGTC
heavy chain primer R (SEQ ID NO: 8):
TAGACCTTTGGAGGAGTTGTGCTAGC CAAGGACACG GTGACCGGGA
the PCR reaction procedure is shown in Table 1.
TABLE 1
The PCR product was recovered by 1% agarose gel. Splicing the amplified light chain variable region and heavy chain variable region into scFv by using an overlap-PCR method, and recovering and storing the products at-20 ℃ by 1% agarose gel.
Example 3: construction and screening of phage Single-chain antibody library
The phagemid vector pcomb3XSS and the ScFv fragment recovered by purification are subjected to enzyme digestion by using SfiI to construct a recombinant plasmid, the competent cells of TG1 are electrically transformed by the recombinant plasmid to construct a rabbit-derived immune single-chain antibody library, and a primary phage single-chain antibody library is prepared; the primary phage single-chain antibody library is enriched and screened for 3 rounds to obtain a specific phage single-chain antibody library with high affinity and strong specificity; selecting monoclonal to prepare monoclonal Phage supernatant, and identifying positive clones by using a Phage-ELISA method to obtain positive sequences.
Example 4: construction, expression and antibody purification of stable transgenic cell lines
1. Construction of stable transgenic cell lines
The heavy chain constant region and the variable region as well as the light chain constant region and the variable region were linked by overlay-PCR, respectively, to obtain recombinant antibody genes. The heavy chain antibody gene and the light chain antibody gene are respectively connected with an expression vector pCHO 1.0 after double digestion of XmaJI/BstZ17I and EcoRV/PacI. After the recombined plasmid is transferred into competent DH5 alpha cells, positive clone sequencing is selected and plasmid extraction is carried out. The extracted plasmid was subjected to RruI enzyme linearization treatment and transfected into CHO cells.
After 48h transfection, ELISA detection antibody is added into a culture medium after expression, 200nmol/L MTX and 20 mu g/mL Puromycin are added for screening positive cell strains, after the cell viability is recovered, the screening concentration is continuously increased (1000 nmol/L MTX and 50 mu g/mL Puromycin), and after the cell viability is recovered, the construction of stable cell strain pool is completed. Then, the stable cell strain pool is subjected to shaking flask of 96-24-6-SF 125, and monoclonal antibody cell strains capable of stably and highly expressing are screened.
2. Recombinant antibody expression
Resuscitate stable high-expression monoclonal cell line according to 5×10 5 After the cell/mL density is transferred for 2 generations, the Fed-batch evaluation is started when the cell activity rate is not lower than 95%, the feeding is added every other day, and the target protein supernatant is obtained after the cell activity rate is harvested below 70%.
3. Identification of column equilibrium by SPA purification and SDS-PAGE of antibodies
Setting the flow rate to be 6.4mL/min, replacing the balance buffer solution to be 0.02mol/L PBS, flushing the chromatographic column at the pH of 7.4, setting the flow rate to be 3.8mL/min, carrying out sample loading and purification, putting a 10 mu L flow through tube into a 250mL conical flask to collect and flow through when the sample in the 10 mu L flow through tube is added into 200 mu L G250 dye liquor to detect bluing, and balancing: setting the flow rate to be 6.4mL/min, and flushing the chromatographic column again with 0.02mol/L PBS (phosphate buffer solution) and pH 7.4 until no protein flows through; dissociation: 10 4mL centrifuge tubes are placed on a dissociation tube rack, 200 mu L of 1mol/L Tris pH 8.5 is added into each tube, a constant flow pump is arranged to set the flow speed to be 6.4mL/min, dissociation buffer solution is used for dissociating target protein, 10 mu L of an equal flow penetrating tube sample is added into 200 mu L G250 dye liquor to detect blue change, manual collection is started, 4mL of each tube is collected, 10 mu L of the equal flow penetrating tube sample is added into 200 mu L G250 dye liquor to detect colorless, and collection is stopped. The collected proteins were pooled and detected by SDS-PAGE.
Example 5: fragmented antibody preparation and purification
In the structure of antibodies, for example, igG, antibodies can be divided into two parts, F (ab ') 2 and Fc, by proteolytic action, F (ab') 2 binding to two antigen binding sites with disulfide bonds and having a molecular weight of about 110kDa. Because the F (ab') 2 fragment removes the Fc part, detection can not be interfered by anti-Fc antibody, and the detection has higher sensitivity. The following method is described by taking pepsin as an example:
1. antibody preparation: 100mg of qualified antibody is taken and placed in a dialysis bag, and is placed in a dialysis solution (single dialysis amount is 1L) with pH of 20mmol/L NaAc of 4.2, and is subjected to standing dialysis at 4 ℃ for 3 times, 2 times of dialysis, 3 hours of first dialysis, 3 hours of second dialysis and 15 hours-16 hours of third dialysis.
2. Reagent preparation: the Pepsin enzyme is restored to room temperature half an hour in advance, is placed in purified water for dialysis, and is subjected to standing treatment for 0.5 hour and then is replaced by the purified water once.
3. And (3) enzyme cutting: pepsin enzyme was weighed according to Pepsin enzyme/antibody mass=1/50 and dissolved by adding 0.7ml 20mmol/L NaAc pH 4.2. 2mL of solubilized Pepsin enzyme was added to the antibody. The reaction cup was placed in a shaker for cleavage reaction (37 ℃, 150r/min, shaking reaction overnight).
4. And (3) terminating: after the reaction, the pH was adjusted to 8.0 with 1mol/L Tris, and the reaction was terminated for 1 to 2 hours with stirring. The enzyme digestion product is placed in 0.02mol/L PBS buffer solution with pH of 7.4 (single dialysis amount is 1L), kept still and dialyzed overnight at 4 ℃, dialyzed 3 times, exchanged 2 times during the dialysis, dialyzed 3 hours for the first time, dialyzed 3 hours for the second time, dialyzed 15 hours to 16 hours for the third time.
5. Sample treatment: and (3) performing fine purification on the enzyme-digested product by using an SPA purification process, collecting the flow-through components, concentrating, and replacing PBS buffer solution for standby. SDS-PAGE analysis was performed on the final product.
Example 6: kit for detecting T4 antigen by using fragmented antibodies prepared by the invention
1. Clinical relevance: the kit constructed by self-produced antibodies is compared with the clinical detection result of the commercial Roche FT4 kit. The self-production kit is evaluated by using an ampere-graph instrument magnetic particle system; the same sample was tested in parallel on a rogowski kit and instrument.
2. Stability (safety image instrument, magnetic particle system evaluation): stability assessment has three main uses: firstly, the kit is used for evaluating the validity period of the kit in the process of transportation, storage and use; secondly, the storage life of the kit is estimated by using an acceleration experiment; thirdly, the stability difference before and after a certain change (such as materials, formulas and the like) is compared. The antibody fraction was accelerated at 37 degrees celsius for 7 days for analytical comparison.
3. Analytical specificity (Anemark instrument, magnetic particle system evaluation): including interfering substances and cross-over substances. Cross material: cross or structural analogues which are known to have partial structural identity or similarity to the present marker, or other substances which have strong homology to the present marker, or substances which are relatively close to the pathogen species.
Effect example
The results of the antiserum titer test in example 1 showed that three sheep were immunized in parallel with the same immunogen and that there was a difference in serum titers after 3-immunization, with the highest titer in sheep # 3 (see Table 2). Phage library screening is preferably performed at high titer 3 #. The results of gel electrophoresis of total RNA of spleen tissue are shown in FIG. 1.
TABLE 2
| Serum dilution ratio | 1# | 2# | 3# | Negative control |
| 1/200 | 472715 | 248953 | 538842 | 2698 |
| 1/1k | 371986 | 149813 | 456964 | 2759 |
| 1/2k | 295630 | 104814 | 369559 | 1961 |
| 1/4k | 222788 | 67973 | 299782 | 2013 |
| 1/8k | 166934 | 44912 | 226009 | 1575 |
| 1/16k | 120255 | 27554 | 166567 | 112 |
| 1/32k | 80192 | 16225 | 110969 | 19 |
| 1/64k | 48632 | 8572 | 71937 | 27 |
(II) in example 2, the results of gel electrophoresis of the amplified products of the light chain variable region and the heavy chain variable region are shown in FIG. 2. The results of gel electrophoresis of the overlap-PCR amplification products of scFv are shown in FIG. 3.
As shown in FIG. 4, the SDS-PAGE results of example 4 show that purity of the purified SPA product can reach 95% or more.
(IV) SDS-PAGE results of example 5 are shown in FIG. 5, and the final product after cleavage and purification by protease was analyzed by SDS-PAGE, and it was judged that cleavage was sufficient by increasing the comparison of the full-length antibody.
(fifth) in example 6:
1) Experimental results of clinical relevance show that the relevance R of a kit for self-producing fragmented antibody combination and a marketed Roche kit is obtained by clinically examining samples in a linear range 2 > 0.98 as shown in figure 6.
2) The experimental results of the stability evaluation show that the signal value and the concentration value have amplitude within 10 percent and have good stability as shown in table 3.
TABLE 3 Table 3
3) As shown in Table 4, the specificity experiment shows that the crossing rate of the self-produced goat monoclonal antibody and rT3 is reduced by 8 times compared with that of the commercial goat monoclonal antibody; the crossing rate of tetraiodothyroacetic acid is reduced by 5.3 times compared with that of the commercial sheep monoclonal antibody; the crossing rate of 3,5' -2 iodine-thyroxine is reduced by 41 times compared with that of the commercial sheep monoclonal antibody. The segmented antibody provided by the invention has better specificity.
TABLE 4 Table 4
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. An antibody, comprising:
the light chain variable region thereof has:
(1) An amino acid sequence shown as SEQ ID NO. 1; or (b)
(2) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (1), and having the same or similar functions as those of (1); or (b)
(3) An amino acid sequence having at least 70% homology with the amino acid sequence as set forth in (1) or (2);
the heavy chain variable region thereof has:
(4) An amino acid sequence shown as SEQ ID NO. 2; or (b)
(5) An amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence shown in (4), and having the same or similar functions as those of (4); or (b)
(6) An amino acid sequence having at least 70% homology with the amino acid sequence as shown in (4) or (5);
the plurality is 2 to 30.
2. A nucleic acid molecule encoding the antibody of claim 1.
3. The nucleic acid molecule of claim 2, comprising:
the device comprises:
(7) A nucleotide sequence shown as SEQ ID NO. 3; or (b)
(8) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (7), and having the same or similar function as (7); or (b)
(9) A nucleotide sequence having at least 70% homology with the nucleotide sequence as set forth in (7) or (8);
and/or
The device comprises:
(10) A nucleotide sequence shown as SEQ ID NO. 4; or (b)
(11) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (10), and having the same or similar function as (10); or (b)
(12) A nucleotide sequence having at least 70% homology with the nucleotide sequence as set forth in (10) or (11);
the plurality is 2 to 90.
4. An expression vector comprising the nucleic acid molecule of claim 2 or 3, and an acceptable genetic element.
5. A host cell comprising the nucleic acid molecule of claim 2 or 3 or the expression vector of claim 4.
6. The method of producing an antibody according to claim 1, comprising:
(A) Ligating the nucleic acid molecule of claim 2 or 3 to a scaffold to obtain an expression vector, introducing the expression vector into a host cell, and culturing to obtain the antibody; or (b)
(B) Introducing the expression vector of claim 4 into a host cell, and culturing to obtain the antibody; or (b)
(C) Culturing the host cell of claim 5 to obtain the antibody.
7. Use of the antibody of claim 1 for the preparation of a reagent or kit for detecting thyroxine.
8. An agent comprising the antibody of claim 1, and an acceptable adjuvant or adjuvant.
9. Kit comprising an antibody according to claim 1 or a reagent according to claim 8, together with acceptable adjuvants or auxiliaries.
10. A device comprising a coated antibody according to claim 1, and an acceptable component.
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