CN110878124A - Antibody fragment of DNA polymerase, antibody and application thereof - Google Patents
Antibody fragment of DNA polymerase, antibody and application thereof Download PDFInfo
- Publication number
- CN110878124A CN110878124A CN201811031800.4A CN201811031800A CN110878124A CN 110878124 A CN110878124 A CN 110878124A CN 201811031800 A CN201811031800 A CN 201811031800A CN 110878124 A CN110878124 A CN 110878124A
- Authority
- CN
- China
- Prior art keywords
- acid sequence
- variable region
- amino acid
- chain variable
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
An antibody fragment of DNA polymerase, an antibody and application thereof, wherein the antibody comprises a heavy chain variable region amino acid sequence which has a sequence shown in any one of SEQ ID NO 1-3; a light chain variable region amino acid sequence having a sequence as shown in any one of SEQ ID NOs 7-9. The monoclonal antibody of the DNA polymerase has the characteristic of high affinity with antigen DNA polymerase, and can specifically neutralize the polymerization of the DNA polymerase.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody fragment of DNA polymerase, an antibody and application thereof.
Background
In 1956 A.Kornberg first found Escherichia coli DNA polymerase I. From this point on, the development and application of DNA polymerase is started. Among them, thermostable DNA polymerases are most widely used in PCR technology. When the DNA polymerase is applied to PCR, the enzyme activity of the DNA polymerase is still remained before the thermal denaturation step, so that the non-specific amplification of PCR is caused, and finally, the sensitivity and the yield of the reaction are reduced. Therefore, methods for reducing adverse reactions are sought.
Several methods currently in use include physical exclusion, chemical modification, and hot-start antibody methods. Among them, the hot start antibody method is currently the most widely used and effective method. The hot start products currently in use include taq hot start DNA polymerase, KOD hot start DNA polymerase and HiFi hot start DNA polymerase.
However, no hot start antibody against pfu DNA polymerase exists at present, and the hot start can only be performed by a physical method in the normal PCR process, so that the use process is complicated, and the application is limited.
Disclosure of Invention
The invention provides an antibody fragment of DNA polymerase, an antibody and application thereof, which can be used for hot start of the DNA polymerase.
According to a first aspect, there is provided in one embodiment an antibody fragment of a DNA polymerase comprising a monoclonal antibody heavy chain variable region amino acid sequence having the sequence set forth in any one of SEQ ID NOs 1 to 3.
According to a second aspect, there is provided in one embodiment an antibody fragment of a DNA polymerase comprising a monoclonal antibody light chain variable amino acid sequence having the sequence set forth in any one of SEQ ID NOs 7-9.
According to a third aspect, one embodiment provides an antibody to a DNA polymerase comprising a heavy chain variable region amino acid sequence having a sequence as set forth in any one of SEQ ID NOs 1-3 and a light chain variable region amino acid sequence; the light chain variable region amino acid sequence has a sequence shown in any one of SEQ ID NO 7-9;
preferably, the amino acid sequence of the heavy chain variable region has the sequence shown in SEQ ID NO 1; the light chain variable region amino acid sequence has a sequence shown as SEQ ID NO. 7;
preferably, the amino acid sequence of the heavy chain variable region has the sequence shown in SEQ ID NO 2; the amino acid sequence of the light chain variable region has a sequence shown as SEQ ID NO. 8;
preferably, the amino acid sequence of the heavy chain variable region has the sequence shown in SEQ ID NO. 3; the amino acid sequence of the light chain variable region has a sequence shown in SEQ ID NO. 9.
According to a fourth aspect, one embodiment provides a nucleic acid sequence encoding a heavy chain variable region amino acid sequence of a monoclonal antibody, comprising a nucleotide sequence encoding the heavy chain variable region amino acid sequence set forth in any one of SEQ ID NOs 1-3;
preferably, the nucleic acid sequence has a sequence as shown in any one of SEQ ID NO 4-6.
According to a fifth aspect, there is provided in one embodiment a nucleic acid sequence encoding a monoclonal antibody light chain variable region amino acid sequence, comprising a nucleotide sequence encoding a light chain variable region amino acid sequence set forth in any one of SEQ ID NOs 7-9;
preferably, the nucleic acid sequence has a sequence as shown in any one of SEQ ID NO 10-12.
According to a sixth aspect, there is provided in one embodiment a nucleic acid sequence encoding an antibody of a DNA polymerase, comprising nucleotide sequences encoding a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence, wherein the nucleotide sequence encoding the heavy chain variable region amino acid sequence comprises a nucleotide sequence encoding the heavy chain variable region amino acid sequence set forth in any one of SEQ ID NOs 1 to 3, and the nucleotide sequence encoding the light chain variable region amino acid sequence comprises a nucleotide sequence encoding the light chain variable region amino acid sequence set forth in any one of SEQ ID NOs 7 to 9;
preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 1, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 7;
preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 2, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 8;
preferably, the nucleotide sequence encoding the heavy chain variable region amino acid sequence includes a nucleotide sequence encoding the heavy chain variable region amino acid sequence shown in SEQ ID NO. 3, and the nucleotide sequence encoding the light chain variable region amino acid sequence includes a nucleotide sequence encoding the light chain variable region amino acid sequence shown in SEQ ID NO. 9.
According to a seventh aspect, there is provided in one embodiment an expression vector comprising the nucleic acid sequence of any one of the fourth to sixth aspects.
According to an eighth aspect, there is provided in one embodiment a recombinant host cell comprising the expression vector of the seventh aspect.
According to a ninth aspect, there is provided in one embodiment a method of producing an antibody fragment or antibody, the antibody fragment or antibody being expressed by the recombinant host cell of the eighth aspect.
According to a tenth aspect, there is provided in one embodiment a use of an antibody fragment of the first or second aspect or an antibody of the third aspect to selectively neutralize the polymerization activity of a DNA polymerase.
The monoclonal antibody of the DNA polymerase has the characteristic of high affinity with antigen DNA polymerase, and can specifically neutralize the polymerization of the DNA polymerase.
Drawings
FIG. 1 is a flow chart of a process for preparing monoclonal antibodies to DNA polymerase in an embodiment of the present invention;
FIG. 2 shows the result of electrophoretic identification of monoclonal antibodies obtained by expression and purification after recombination in the examples of the present invention;
FIG. 3 shows the result of the identification of the affinity activity of a monoclonal antibody in the examples of the present invention;
FIG. 4 shows the result of identifying the affinity activity of an antigen and an antibody by Western blot experiment in the embodiment of the present invention;
FIG. 5 shows the results of PCR activity assay of hot-start DNA polymerase of monoclonal antibody in the examples of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.
The invention takes DNA polymerase as antigen to immunize a mouse, takes the spleen of the mouse to fuse with myeloma to obtain hybridoma cells, and obtains a monoclonal antibody capable of neutralizing the polymerization activity of the DNA polymerase through activity screening. Then obtaining hybridoma cell RNA through reverse transcription screening, obtaining a gene sequence for coding the monoclonal antibody through PCR, constructing a recombinant vector by the monoclonal antibody sequence through a recombination means, and transfecting CHO cells to obtain a recombinant cell strain. And purifying by an affinity column to obtain the electrophoretically pure monoclonal antibody. The detection of the monoclonal antibody for inhibiting the activity of the DNA polymerase shows that the monoclonal antibody can effectively inhibit the polymerization activity of the DNA polymerase under the conditions of normal temperature or low temperature.
In one embodiment, the present invention provides an antibody fragment of DNA polymerase, comprising a heavy chain variable region amino acid sequence of a monoclonal antibody having a sequence as set forth in any one of SEQ ID NOs 1-3.
SEQ ID NO:1：
EVQLEESGPGLVAPSQSLSLTCTVSGFSLTDYGVNWVRQPPGEGLEWLGMIWVDGSTDYNSVLKSRLIIRKDNSKSQVFLKMNSLQTDDTARYYCARGGYYLDFWGQGTTLTVSSAKTTPPSDYPLA
SEQ ID NO:2：
EVQLEESGPGLVAPSQSLSITCTVSGFSLTTYGVTWVRQPPGKGLEWLGTIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNSLQTDDTATYYCAKYDANVMDYWGQGTSVTVSSAKTTPPSDYPLA
SEQ ID NO:3：
EVQLEESGGGLVKPGGSLKLSCDASGFTFSTYAMSWVRLSPEKRLEWVAEISSGSSYTYFPVTVTGRFTISRDNVKNTLYLEMNSLKSEDTAMYYCVRRDDYGVFAYWGQGTLVTVSAAKTTPPSDYPLA
It is known that the specific binding activity of an antibody is determined by a variable region, and therefore, the present invention defines an antibody sequence by defining an amino acid sequence of a variable region. Having the sequence shown in any one of SEQ ID NOs 1 to 3 in the amino acid sequence of the heavy chain variable region of the monoclonal antibody of the present embodiment means that the amino acid sequence of the heavy chain variable region is the sequence shown in any one of SEQ ID NOs 1 to 3, or may further include other sequences such as a linker (linker) or the like without affecting the specific binding activity of the antibody, and such a linker may be, for example, a sequence in which the amino acid sequence of the heavy chain variable region is linked to the sequence of the constant region.
In the present embodiment, the term "antibody fragment" refers to a portion of an antibody, particularly a monoclonal antibody heavy chain variable region amino acid sequence or a monoclonal antibody light chain variable region amino acid sequence. These antibody fragments have some practical uses, for example, as neutralizers for antigens, for neutralizing antigens, and the like.
In one embodiment, the present invention provides a nucleic acid sequence encoding a heavy chain variable region amino acid sequence of a monoclonal antibody, comprising a nucleotide sequence encoding the heavy chain variable region amino acid sequence set forth in any one of SEQ ID NOs 1-3.
It is to be noted that, based on the principle of base degeneracy, a nucleotide sequence encoding an amino acid sequence of a heavy chain variable region is not the only constant sequence, and any nucleotide sequence capable of encoding the same amino acid sequence of a heavy chain variable region is a nucleic acid sequence within the scope of the present invention.
In a preferred embodiment of the invention, the nucleic acid sequence has the sequence shown in any one of SEQ ID NO 4-6.
SEQ ID NO:4：
TGGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGCTGAGGAGACTGTGAGAGTGGTGCCTTGGCCCCAGAAGTCAAGGTAGTAACCACCTCTGGCACAGTAGTACCTGGCTGTGTCATCAGTTTGTAGACTGTTCATTTTTAAGAAAACTTGGCTCTTGGAGTTGTCCTTCCTGATGATCAGTCTGGATTTGAGAACTGAATTATAGTCTGTACTTCCATCGACCCAAATCATTCCCAGCCACTCCAGACCCTCTCCTGGAGGCTGGCGAACCCAGTTTACACCATAGTCGGTTAATGAGAACCCTGAGACGGTGCATGTGAGGGACAGGCTCTGTGAGGGCGCCACCAGGCCAGGTCCTGACTCCTCCAGCTGCACCTCA
SEQ ID NO:5：
TGGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGCTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATAACATTCGCGTCGTACTTGGCACAGTAGTACGTGGCTGTGTCATCAGTTTGCAGACTGTTCAGTTTTAAGAAAACTTGGCTCTTGGAGTTATCCTTGCTGATGCTCAGTCTGGATATGAGAGCTGAATGATAATTTGTGCTCCCGTCACCCCATATTGTTCCCAGCCACTCCAGACCCTTTCCTGGAGGCTGGCGAACCCAGGTTACACCATAGGTGGTTAATGAGAACCCTGAGACAGTGCATGTGATGGACAGGCTCTGTGAGGGCGCCACCAGGCCAGGTCCTGACTCCTCCAGCTGCACCTCA
SEQ ID NO:6：
TGGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGCTGCAGAGACAGTGACCAGAGTCCCTTGGCCCCAGTAAGCAAACACGCCGTAATCATCCCTCCTCACACAATAATACATGGCCGTGTCCTCAGACTTCAGACTGTTCATTTCCAGGTATAGGGTGTTCTTGACATTGTCCCTGGAGATGGTGAATCGGCCCGTCACAGTGACTGGAAAGTAGGTGTAACTACTACCACTACTAATTTCTGCGACCCACTCCAGCCTCTTCTCTGGAGACAGGCGAACCCAAGACATGGCATAGGTACTGAAAGTGAATCCAGAGGCATCACAGGAGAGTTTCAGGGACCCTCCAGGCTTCACTAAGCCTCCCCCAGACTCCTcCAGCTGCACCTCA
Wherein, the nucleic acid sequence SEQ ID NO. 4 encodes the heavy chain variable region amino acid sequence shown in SEQ ID NO. 1; the nucleic acid sequence SEQ ID NO. 5 encodes the heavy chain variable region amino acid sequence shown in SEQ ID NO. 2; the nucleic acid sequence SEQ ID NO 6 encodes the amino acid sequence of the heavy chain variable region shown in SEQ ID NO 3.
In one embodiment, the present invention provides an antibody fragment of DNA polymerase, comprising a monoclonal antibody light chain variable region amino acid sequence having a sequence as set forth in any one of SEQ ID NOS 7-9.
SEQ ID NO:7：
DIQLTQSPSSMYASLGERVTITCKASQDIKTYISWYQQKPWKSPKTLIYYATTLADGVPSRFSGSGSGQDYSLTISSLESDDIATYYCLQQYESPFTFGSGTKLDIKRADAAPTVS
SEQ ID NO:8：
DILRTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSTPLTFGGGTKLEIKRADAAPTVS
SEQ ID NO:9：
DIQLTQSPASLSAPVGETVTITCRASEIIYSYLAWYLQKQGKSPHLLVYNAKTLAEGVPSRFSGSGSGTQFYLRINNLQPEDFGIYYCQHHYGRPWTFGGGTNLEIKGADAAPTVS
It is known that the specific binding activity of an antibody is determined by a variable region, and therefore, the present invention defines an antibody sequence by defining an amino acid sequence of a variable region. Having the sequence shown in any one of SEQ ID NOs 7 to 9 in the light chain variable region amino acid sequence of the monoclonal antibody of the present embodiment means that the light chain variable region amino acid sequence is the sequence shown in any one of SEQ ID NOs 7 to 9, or may further include other sequences such as a linker sequence (linker) or the like without affecting the specific binding activity of the antibody, and such a linker sequence may be, for example, a sequence in which the light chain variable region amino acid sequence is linked to the constant region sequence.
In one embodiment, the present invention provides a nucleic acid sequence encoding a light chain variable region amino acid sequence of a monoclonal antibody, comprising a nucleotide sequence encoding the light chain variable region amino acid sequence set forth in any one of SEQ ID NOs 7-9.
It is to be noted that, based on the base degeneracy principle, the nucleotide sequence encoding the amino acid sequence of the light chain variable region is not the only constant sequence, and any nucleotide sequence capable of encoding the same amino acid sequence of the light chain variable region is a nucleic acid sequence within the scope of the present invention.
In a preferred embodiment of the invention, the nucleic acid sequence has the sequence shown in any one of SEQ ID NO 10-12.
SEQ ID NO:10：
TGGATACAGTTGGTGCAGCATCAGCCCGTTTTATGTCCAACTTTGTCCCCGAGCCGAACGTGAATGGGCTCTCATACTGTTGTAGACAGTAATAAGTTGCTATATCGTCAGACTCCAGGCTGCTGATGGTTAGAGAATAATCTTGCCCAGATCCACTGCCACTGAATCTTGATGGGACCCCATCCGCCAAGGTTGTTGCATAATAGATCAGGGTCTTAGGAGATTTCCATGGTTTCTGCTGGTACCAGCTTATATAGGTTTTAATGTCCTGACTCGCCTTGCAAGTGATAGTGACTCTCTCTCCCAGCGATGCATACATGGAGGATGGAGACTGAGTCAGCTGGATGTCASEQ ID NO:11：
TGGATACAGTTGGTGCAGCATCAGCCCGTTTGATTTCCAGCTTGGTGCCTCCACCGAACGTCAGAGGAGTACTGTAAAGTTGTTGACAGTAATAACTTACAAAATCTTCAGCCTGTAGGCTGCTGATCTTGAAAGAAAATTTTGTGCCAGATCCACTACCACTGAACCTTGATGGGACCCCATCTGCCAAGCTGGTTGCAGCATAAATCAGGAGCTGAGGAGATTTCCCTGGTTTCTGCTGATACCATGCTAACCATGTACCAATGGTCTGACTTGCCAGGCATGTGATGGTGACACTTTCTCCCAGAGATGCAGACTGGGAGGCAGGAGACTGGGTCCTCAGAATGTCASEQ ID NO:12：
TGACATCCAGCTGACTCAGTCTCCAGCCTCCCTATCTGCACCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGATTATTTACAGTTATTTAGCATGGTATCTACAGAAACAGGGAAAATCTCCTCACCTCTTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTATCTGAGGATCAACAACCTGCAGCCTGAGGATTTTGGGATTTATTACTGTCAACATCATTATGGTCGTCCGTGGACGTTCGGTGGAGGCACCAACCTGGAAATCAAAGGGGCTGATGCTGCACCAACTGTATCCA
Wherein, the nucleic acid sequence SEQ ID NO. 10 encodes the heavy chain variable region amino acid sequence shown in SEQ ID NO. 7; the nucleic acid sequence SEQ ID NO. 11 encodes the heavy chain variable region amino acid sequence shown in SEQ ID NO. 8; the nucleic acid sequence SEQ ID NO. 12 encodes the heavy chain variable region amino acid sequence shown in SEQ ID NO. 9.
In one embodiment, the present invention provides an antibody to a DNA polymerase, comprising a heavy chain variable region amino acid sequence having a sequence as set forth in any one of SEQ ID NOS 1-3; the light chain variable region amino acid sequence has a sequence shown in any one of SEQ ID NO 7-9.
In a preferred embodiment of the present invention, the heavy chain variable region amino acid sequence has the sequence shown in SEQ ID NO. 1; the variable region amino acid sequence of the light chain has the sequence shown in SEQ ID NO. 7. In a preferred embodiment of the present invention, the heavy chain variable region amino acid sequence has the sequence shown in SEQ ID NO. 2; the variable region amino acid sequence of the light chain has the sequence shown in SEQ ID NO. 8. In a preferred embodiment of the present invention, the heavy chain variable region amino acid sequence has the sequence shown in SEQ ID No. 3; the variable region amino acid sequence of the light chain has the sequence shown in SEQ ID NO 9.
In the present embodiment, the "antibody" is structurally the same as a normal IgG antibody, i.e., comprises two heavy chains and two light chains, wherein the heavy chains comprise heavy chain variable regions and heavy chain constant regions, and the light chains comprise light chain variable regions and constant regions, and wherein the heavy chain constant regions and light chain variable regions are not different from those of a normal IgG antibody.
In one embodiment of the present invention, there is provided a nucleic acid sequence encoding an antibody to DNA polymerase, comprising a nucleotide sequence encoding a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence, wherein the nucleotide sequence encoding the heavy chain variable region amino acid sequence comprises a nucleotide sequence encoding the heavy chain variable region amino acid sequence set forth in any one of SEQ ID NOs 1 to 3, and the nucleotide sequence encoding the light chain variable region amino acid sequence comprises a nucleotide sequence encoding the light chain variable region amino acid sequence set forth in any one of SEQ ID NOs 7 to 9.
In a preferred embodiment of the present invention, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 1, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises the nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 7. In a preferred embodiment of the present invention, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 2, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises the nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 8. In a preferred embodiment of the present invention, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises the nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 9.
In one embodiment of the invention, an expression vector is provided comprising a nucleic acid sequence encoding an antibody fragment or antibody of the invention. The vector backbone of the present invention is not particularly limited, and those skilled in the art can obtain the expression vector of the present invention by ligating a nucleic acid sequence encoding the antibody fragment or antibody of the present invention into an appropriate vector backbone as required. In one embodiment of the invention, the vector backbone is pcdna3.1.
In one embodiment of the invention, a recombinant host cell is provided comprising an expression vector of the invention. The host cell of the present invention is not particularly limited, and one skilled in the art can introduce an expression vector into a suitable host cell as needed for expressing the antibody fragment or antibody of the present invention.
In one embodiment of the invention, a method of producing an antibody fragment or antibody is provided, the antibody fragment or antibody being expressed by a recombinant host cell of the invention.
In one embodiment of the invention there is provided the use of an antibody fragment or antibody of the invention to selectively neutralise the polymerisation activity of a DNA polymerase.
The monoclonal antibody of the DNA polymerase has the characteristic of high affinity with antigen DNA polymerase, and can specifically neutralize the polymerization of the DNA polymerase.
The technical solutions of the present invention are described in detail below by way of examples, and it should be understood that the examples are only illustrative and should not be construed as limiting the scope of the present invention.
FIG. 1 shows a process flow for preparing an antibody against DNA polymerase in the following examples.
Example 1 immunization of mice
Taking pfu DNA polymerase as an antigen, dissolving the antigen in PBS, carrying out intraperitoneal injection on a C57 mouse by using an emulsified liquid containing 25-100 mu g of the antigen and Freund's complete adjuvant in the PBS with the same volume, carrying out mouse immunization, and carrying out ELISA detection on serum after three times of conventional immunization. Antigen was coated on enzyme strips, serum dilutions incubated with anti-mouse IgG with HRP and finally developed with TMB. After detection, mice 2, 4 and 5 with higher titer are selected for the next step of experiment.
Table 1 shows the results of the ELISA test of the serum titers of immunized mice, which describes the antibody titer produced by each mouse after the mice were immunized with the antigen.
TABLE 1
Example 2 cell fusion
1. Preparation of myeloma cells
In this example, SP2/0Ag14 myeloma cells were used, the cells were cultured to a good growth state and a good morphology, and the cells were cultured to a logarithmic growth phase before fusion, and suspended to prepare a suspension with a certain cell density.
2. Preparation of splenocytes
The mice with the highest titer were selected and sacrificed. Fixing the dissected plate, taking out spleen, placing in fresh culture medium, removing adhesive tissue, grinding spleen, filtering with 200 mesh sun screen, washing, adding erythrocyte lysate for lysis for 10min, adding fresh culture medium for dilution, centrifuging to remove supernatant, dissolving in culture medium again, measuring cell density, and diluting cells to a certain concentration.
3. Cell fusion
The SP2/0Ag14 cells were mixed with spleen cells at a ratio of 1:10, and then fused with polyethylene glycol 2000(PEG2000) to obtain hybridoma cells, which were suspended in HAT medium containing feeder cells and pressurized, and then added to a 96-well plate for culture. Hybridoma mother clones were obtained.
Example 3 hybridoma subclone screening
After culturing the hybridoma cells in the 96-well plate of example 2 for 2 weeks, the supernatants were subjected to ELISA to identify the positive antibody values in the supernatants. Screening to obtain positive cell strains, and performing secondary limiting dilution subcloning. Then ELISA is carried out to identify and screen positive cell strains. The ELISA screening procedure was as follows:
(1) 100 μ l of antigen (DNA polymerase concentration 1 μ g/ml) was coated onto the enzyme strips overnight. Wash with 200 μ l PBST for 10min with shaking, 3 times, wash with PBS 3 times with shaking.
(2) Add 1% BSA-PBS 200. mu.l, block for 2h at 37 ℃. PBST200 u l, 10min, washing 3 times, PBS200 u l, 10min, washing 3 times.
(3) Mu.l of diluted supernatant was added and incubated at 37 ℃ for 2 h. PBST200 u l, 10min, washing 3 times, PBS200 u l, 10min, washing 3 times.
(4) Diluted 100. mu.l of a commercial horseradish peroxidase-conjugated goat anti-mouse antibody was added and incubated at 37 ℃ for 1 h. PBST200 u l, 10min, washing 3 times, PBS200 u l, 10min, washing 3 times.
(5) Color development was performed for 10min using tetramethylbenzidine microporous peroxidase substrate (TMB) as substrate.
(6) After the reaction was terminated by adding 50. mu.l of sulfuric acid having a concentration of 0.1mol/L, absorbance at 450nm was measured.
Table 2 shows the results of ELISA positive detection of the monoclonal antibody, and describes the OD450 values of ELISA detection of the ELISA-selected positive clones.
TABLE 2
Example 4 monoclonal antibody preparation and neutralization Activity screening
And (3) carrying out expanded culture on the positive hybridoma cell strain. The supernatant was collected, centrifuged at 8000rpm for 15min, the sample was filtered through a 0.22 μm membrane and purified using a protein A (protein A) column, and the eluate was used for elution to obtain the purified monoclonal antibody. The monoclonal antibodies were screened using a polymerase polymerization activity identification assay. Finally, the monoclonal antibody capable of inhibiting the polymerization activity of the DNA polymerase is obtained by screening. The screening experiment for inhibiting the polymerase activity by the monoclonal antibody is as follows:
(1) monoclonal antibodies were mixed with DNA polymerase in a molecular ratio of 1:1 and incubated at room temperature for 2 hours.
(2) The reaction was carried out according to the following system of Table 3:
TABLE 3
The reaction was carried out at 37 ℃ for 2 hours.
And (3) identifying the double-stranded DNA content of the product by using a fluorescent dye method, and further screening the effect of the monoclonal antibody on inhibiting the polymerization activity. The fluorescent agent is prepared by taking out 2 μ l of reaction solution, adding diluted fluorescent dye, and detecting with a calibrated Qubit instrument.
Table 4 shows the results of screening experiments for the inhibition of polymerase activity by monoclonal antibodies, depicting the results of inhibition of the polymerization activity of DNA polymerase by positive monoclonal antibodies. From the results, three cell lines of monoclonal antibodies 7H7, 8D9 and 12E12 with good inhibition effect can be screened.
TABLE 4
EXAMPLE 5 obtaining of monoclonal antibody sequences
The method comprises the steps of obtaining a cDNA library of hybridoma cells by extracting RNA of the screened monoclonal antibody hybridoma cells with effective effects and carrying out reverse transcription, designing degenerate primers according to known murine antibody heavy and light chain constant regions, carrying out PCR on the cDNA by using the designed primers to obtain monoclonal antibody heavy chain and light chain variable region sequences, then introducing the variable region sequences into a T vector by cloning, introducing into escherichia coli DH5 α for amplification, selecting clones, carrying out expanded culture, extracting a recombinant vector, and carrying out sequencing to obtain antibody heavy and light chain variable region sequences.
Wherein, the amino acid sequence of the heavy chain variable region of the 7H7 antibody is shown as SEQ ID NO. 1, and the coding nucleic acid sequence thereof is shown as SEQ ID NO. 4; the variable region of light chain has the amino acid sequence as shown in SEQ ID No. 7 and the coding nucleic acid sequence as shown in SEQ ID No. 10. The amino acid sequence of the heavy chain variable region of the 8D9 antibody is shown as SEQ ID NO. 2, and the coding nucleic acid sequence thereof is shown as SEQ ID NO. 5; the variable region amino acid sequence of the light chain is shown as SEQ ID NO. 8, and the coding nucleic acid sequence is shown as SEQ ID NO. 11. The amino acid sequence of the heavy chain variable region of the 12E12 antibody is shown as SEQ ID NO. 3, and the coding nucleic acid sequence thereof is shown as SEQ ID NO. 6; the variable region amino acid sequence of the light chain is shown as SEQ ID NO. 9, and the coding nucleic acid sequence is shown as SEQ ID NO. 12.
Example 6 construction of monoclonal antibody expression vector
Connecting the variable region sequences of the heavy and light chains of the obtained antibody with a constant region of a murine antibody to obtain a complete full-length antibody sequence. And then the obtained heavy-light chain is connected with a eukaryotic expression vector pCDNA3.1 through double enzyme digestion enzyme connection to obtain a recombinant expression vector H-pCDNA3.1 and a recombinant expression vector L-pCDNA3.1.
Typical but non-limiting examples of murine constant regions in this example are the IgG1 type murine heavy chain constant region and the murine kappa light chain constant region, the sequences of which are as follows:
heavy chain constant region CH:
Vprdcgckpcictvpevssvfifppkpkdvltitltpkvtcvvvdiskddpevqfswfvddvevhtaqtqpreeqfnstfrsvselpimhqdwlngkefkcrvnsaafpapiektisktkgrpkapqvytipppkeqmakdkvsltcmitdffpeditvewqwngqpaenykntqpimntngsyfvysklnvqksnweagntftcsvlheglhnhhtekslshspglqldetcaeaqdgeldglwtt(SEQ ID NO：13)。
light chain constant region CL:
APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC(SEQ ID NO：14)。
EXAMPLE 7 expression purification and characterization of monoclonal antibodies
The recombinant plasmids H-pCDNA3.1 and L-pCDNA3.1 are electrically transferred into a CHO cell strain, and single clone is picked through three rounds to obtain a high-yield cell strain for stably expressing the fusion protein. And (3) carrying out amplification culture on the high-yield cell strain, taking the supernatant, centrifuging at 8000rpm for 15min, purifying a filter membrane filtration sample with a 0.22 mu m membrane by using a protein A (protein A) column, and eluting by using an eluent to obtain the purified monoclonal antibody. Samples were identified using SDS-PAGE.
FIG. 2 shows the result of electrophoretic identification of the monoclonal antibody obtained by expression purification after recombination, wherein, lane 1 is non-reduction electrophoresis of monoclonal antibody 7H 7; lane 2 non-reducing electrophoresis of monoclonal antibody 8D 9; lane 3 non-reducing electrophoresis of monoclonal antibody 12E 12; lane M protein marker; lane 4 monoclonal antibody 7H7 reduction electrophoresis; lane 5 reduction electrophoresis with monoclonal antibody 8D 9; lane 6, monoclonal antibody 12E12 reduction electrophoresis.
EXAMPLE 8 monoclonal antibody Activity identification experiment
1. The ELISA assay identified the affinity activity of the preferred 3 monoclonal antibodies (7H7, 8D9, and 12E 12).
(1) 100 μ l of antigen (DNA polymerase concentration 1 μ g/ml) was coated onto the enzyme strips overnight. Wash with 200 μ l PBST for 10min with shaking, 3 times, wash with PBS 3 times with shaking.
(2) Add 1% BSA-PBS 200. mu.l, block for 2h at 37 ℃. PBST200 u l, 10min, washing 3 times, PBS200 u l, 10min, washing 3 times.
(3) The antibody was diluted at the following concentrations, 100. mu.l of the diluted supernatant was added, and incubated at 37 ℃ for 2 hours. PBST200 u l, 10min, washing 3 times, PBS200 u l, 10min, washing 3 times.
(4) Diluted 100. mu.l of a commercial horseradish peroxidase-conjugated goat anti-mouse antibody was added and incubated at 37 ℃ for 1 h. PBST200 u l, 10min, washing 3 times, PBS200 u l, 10min, washing 3 times.
(5) Color development was performed for 10min using tetramethylbenzidine microporous peroxidase substrate (TMB) as substrate.
(6) After the reaction was terminated by adding 50. mu.l of sulfuric acid having a concentration of 0.1mol/l, absorbance at 450nm was measured.
FIG. 3 shows the result of identifying the affinity activity of a monoclonal antibody, showing that the ELISA experiment identifies the affinity activity of a monoclonal antibody to an antigen (DNA polymerase).
2. Western blot experiment for identifying affinity activity of monoclonal antibody and antigen
And carrying out Western Blot affinity activity identification on the obtained high-purity monoclonal antibody sample. Electrophoresis of antigen DNA polymerase, after electrophoresis, constant current transfer at 4 ℃ and 250mA for 1.5h, protein was transferred to PVDF membrane (purchased from Millipore); after the transfer, the membrane was blocked in 1% BSA-TBS at 37 ℃ for 2 h; washing with PBS for 3 times, adding HRP-labeled goat anti-mouse IgG polyclonal antibody at a ratio of 1: 2000, incubating at 37 deg.C for 1h, washing with TBS for 3 times, adding ECL luminescence developing solution dropwise, and exposing with gel imager for photographing.
FIG. 4 shows the result of identifying the affinity activity of the antigen and antibody by Western blot, which shows that the binding of the monoclonal antibody to the antigen (DNA polymerase) at molecular level is identified by Western blot, wherein lanes 1-3 sequentially show the binding of the antibodies 7H7, 8D9 and 12E12 to the antigen (DNA polymerase) at molecular level.
3. Monoclonal antibody hot start DNA polymerase PCR activity identification experiment
(1) The antibodies were mixed with DNA polymerase in proportion and divided into 4 groups, the first group being monoclonal antibody 7H7+ DNA polymerase: 1: 1; the second group was monoclonal antibody 8D9+ DNA polymerase: 1: 1; the third group is monoclonal antibody 12E12+ DNA polymerase: 1: 1; the fourth group was monoclonal antibody 7H7+8D9+12E12+ DNA polymerase: 1:1:1: 1; the fifth group is DNA polymerases.
(2) The template is lambda DNA,
the primers are F: CCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGG (SEQ ID NO: 15) and
the primer is R: CGGGAATACGACGGTTACCCACCACAAGCACG (SEQ ID NO: 16).
The destination stripe size is 8 KD.
(3) The reaction system was configured as follows:
TABLE 5
The PCR reaction conditions are shown in Table 6 below:
TABLE 6
And (5) carrying out agarose electrophoresis identification after the reaction is finished.
FIG. 5 shows the results of the PCR activity assay for the hot-start DNA polymerase of monoclonal antibody, in lane 1 monoclonal antibody 7H7+ DNA polymerase; lane 2 monoclonal antibody 8D9+ DNA polymerase; lane 3 monoclonal antibody 12E12+ DNA polymerase; lane 4 monoclonal antibody 7H7+8D9+12E12+ DNA polymerase; lane 5: a DNA polymerase; lane M DNAmarker. Shows that the antibody can always polymerize at normal temperature after being mixed with DNA polymerase, thereby improving the yield of the DNA polymerase by hot start PCR.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
SEQUENCE LISTING
<110> Shenzhen Huashengshengsciences institute
<120> antibody fragment of DNA polymerase, antibody and use thereof
<130>18I26830
<160>16
<170>PatentIn version 3.3
<210>1
<211>127
<212>PRT
<213> mice
<400>1
Glu Val Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr
20 25 30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Leu
35 40 45
Gly Met Ile Trp Val Asp Gly Ser Thr Asp Tyr Asn Ser Val Leu Lys
50 55 60
Ser Arg Leu Ile Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Gly Gly Tyr Tyr Leu Asp PheTrp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Asp Tyr Pro Leu Ala
115 120 125
<210>2
<211>128
<212>PRT
<213> mice
<400>2
Glu Val Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr
20 25 30
Gly Val Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Thr Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys Tyr Asp Ala Asn Val Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Asp Tyr Pro Leu Ala
115 120 125
<210>3
<211>130
<212>PRT
<213> mice
<400>3
Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Asp Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Ala Met Ser Trp Val Arg Leu Ser Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Glu Ile Ser Ser Gly Ser Ser Tyr Thr Tyr Phe Pro Val Thr Val
50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Val Arg Arg Asp Asp Tyr Gly Val Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Asp Tyr Pro
115 120 125
Leu Ala
130
<210>4
<211>383
<212>DNA
<213> mice
<400>4
tggccagtgg atagtcagat gggggtgtcg ttttggctga ggagactgtg agagtggtgc 60
cttggcccca gaagtcaagg tagtaaccac ctctggcaca gtagtacctg gctgtgtcat 120
cagtttgtag actgttcatt tttaagaaaa cttggctctt ggagttgtcc ttcctgatga 180
tcagtctgga tttgagaact gaattatagt ctgtacttcc atcgacccaa atcattccca 240
gccactccag accctctcct ggaggctggc gaacccagtt tacaccatag tcggttaatg 300
agaaccctga gacggtgcat gtgagggaca ggctctgtga gggcgccacc aggccaggtc 360
ctgactcctc cagctgcacc tca 383
<210>5
<211>386
<212>DNA
<213> mice
<400>5
tggccagtgg atagtcagat gggggtgtcg ttttggctga ggagacggtg actgaggttc 60
cttgacccca gtagtccata acattcgcgt cgtacttggc acagtagtac gtggctgtgt 120
catcagtttg cagactgttc agttttaaga aaacttggct cttggagtta tccttgctga 180
tgctcagtct ggatatgaga gctgaatgat aatttgtgct cccgtcaccc catattgttc 240
ccagccactc cagacccttt cctggaggct ggcgaaccca ggttacacca taggtggtta 300
atgagaaccc tgagacagtg catgtgatgg acaggctctg tgagggcgcc accaggccag 360
gtcctgactc ctccagctgc acctca 386
<210>6
<211>392
<212>DNA
<213> mice
<400>6
tggccagtgg atagtcagat gggggtgtcg ttttggctgc agagacagtg accagagtcc 60
cttggcccca gtaagcaaac acgccgtaat catccctcct cacacaataa tacatggccg 120
tgtcctcaga cttcagactg ttcatttcca ggtatagggt gttcttgaca ttgtccctgg 180
agatggtgaa tcggcccgtc acagtgactg gaaagtaggt gtaactacta ccactactaa 240
tttctgcgac ccactccagc ctcttctctg gagacaggcg aacccaagac atggcatagg 300
tactgaaagt gaatccagag gcatcacagg agagtttcag ggaccctcca ggcttcacta 360
agcctccccc agactcctcc agctgcacct ca 392
<210>7
<211>116
<212>PRT
<213> mice
<400>7
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Thr Tyr
20 25 30
Ile Ser Trp Tyr Gln Gln Lys Pro Trp Lys Ser Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Asp Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Gln Tyr Glu Ser Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Asp Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser
115
<210>8
<211>116
<212>PRT
<213> mice
<400>8
Asp Ile Leu Arg Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly
1 5 10 15
Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Thr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser
115
<210>9
<211>116
<212>PRT
<213> mice
<400>9
Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Pro Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Ile Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Leu Gln Lys Gln Gly Lys Ser Pro His Leu Leu Val
35 40 45
Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Tyr Leu Arg Ile Asn Asn Leu Gln Pro
65 7075 80
Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His His Tyr Gly Arg Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys Gly Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser
115
<210>10
<211>350
<212>DNA
<213> mice
<400>10
tggatacagt tggtgcagca tcagcccgtt ttatgtccaa ctttgtcccc gagccgaacg 60
tgaatgggct ctcatactgt tgtagacagt aataagttgc tatatcgtca gactccaggc 120
tgctgatggt tagagaataa tcttgcccag atccactgcc actgaatctt gatgggaccc 180
catccgccaa ggttgttgca taatagatca gggtcttagg agatttccat ggtttctgct 240
ggtaccagct tatataggtt ttaatgtcct gactcgcctt gcaagtgata gtgactctct 300
ctcccagcga tgcatacatg gaggatggag actgagtcag ctggatgtca 350
<210>11
<211>350
<212>DNA
<213> mice
<400>11
tggatacagt tggtgcagca tcagcccgtt tgatttccag cttggtgcct ccaccgaacg 60
tcagaggagt actgtaaagt tgttgacagt aataacttac aaaatcttca gcctgtaggc 120
tgctgatctt gaaagaaaat tttgtgccag atccactacc actgaacctt gatgggaccc 180
catctgccaa gctggttgca gcataaatca ggagctgagg agatttccct ggtttctgct 240
gataccatgc taaccatgta ccaatggtct gacttgccag gcatgtgatg gtgacacttt 300
ctcccagaga tgcagactgg gaggcaggag actgggtcct cagaatgtca 350
<210>12
<211>350
<212>DNA
<213> mice
<400>12
tgacatccag ctgactcagt ctccagcctc cctatctgca cctgtgggag aaactgtcac 60
catcacatgt cgagcaagtg agattattta cagttattta gcatggtatc tacagaaaca 120
gggaaaatct cctcacctct tggtctataa tgcaaaaacc ttagcagaag gtgtgccatc 180
aaggttcagt ggcagtggat caggcacaca gttttatctg aggatcaaca acctgcagcc 240
tgaggatttt gggatttatt actgtcaaca tcattatggt cgtccgtgga cgttcggtgg 300
aggcaccaac ctggaaatca aaggggctga tgctgcacca actgtatcca 350
<210>13
<211>247
<212>PRT
<213> mice
<400>13
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu
1 5 10 15
Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr
20 2530
Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys
35 40 45
Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val
50 55 60
His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
65 70 75 80
Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val
115 120 125
Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser
130 135 140
Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu
145 150 155 160
Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro
165 170 175
Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val
180 185190
Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu
195 200 205
His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser
210 215 220
Pro Gly Leu Gln Leu Asp Glu Thr Cys Ala Glu Ala Gln Asp Gly Glu
225 230 235 240
Leu Asp Gly Leu Trp Thr Thr
245
<210>14
<211>103
<212>PRT
<213> mice
<400>14
Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser
1 5 10 15
Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp
20 25 30
Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val
35 40 45
Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met
50 55 60
Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser
65 70 7580
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys
85 90 95
Ser Phe Asn Arg Gly Glu Cys
100
<210>15
<211>35
<212>DNA
<213> Artificial sequence
<400>15
cctctgtcgt ttcctttctc tgtttttgtc cgtgg 35
<210>16
<211>32
<212>DNA
<213> Artificial sequence
<400>16
cgggaatacg acggttaccc accacaagca cg 32
Claims (10)
1. An antibody fragment of DNA polymerase, comprising a heavy chain variable region amino acid sequence of a monoclonal antibody having a sequence set forth in any one of SEQ ID NOs 1-3.
2. An antibody fragment of DNA polymerase, comprising a monoclonal antibody light chain variable region amino acid sequence having a sequence set forth in any one of SEQ ID NOs 7-9.
3. An antibody to a DNA polymerase, comprising a heavy chain variable region amino acid sequence having a sequence set forth in any one of SEQ ID NOS 1-3 and a light chain variable region amino acid sequence; the light chain variable region amino acid sequence has a sequence shown in any one of SEQ ID NO 7-9;
preferably, the heavy chain variable region amino acid sequence has the sequence shown in SEQ ID NO 1; the light chain variable region amino acid sequence has a sequence shown as SEQ ID NO. 7;
preferably, the heavy chain variable region amino acid sequence has the sequence shown in SEQ ID NO 2; the light chain variable region amino acid sequence has a sequence shown as SEQ ID NO. 8;
preferably, the heavy chain variable region amino acid sequence has the sequence shown in SEQ ID NO. 3; the light chain variable region amino acid sequence has a sequence shown in SEQ ID NO. 9.
4. A nucleic acid sequence encoding a heavy chain variable region amino acid sequence of a monoclonal antibody, wherein the nucleic acid sequence comprises a nucleotide sequence encoding the heavy chain variable region amino acid sequence set forth in any one of SEQ ID NOs 1-3;
preferably, the nucleic acid sequence has a sequence as shown in any one of SEQ ID NO 4-6.
5. A nucleic acid sequence encoding a light chain variable region amino acid sequence of a monoclonal antibody, wherein the nucleic acid sequence comprises a nucleotide sequence encoding the light chain variable region amino acid sequence set forth in any one of SEQ ID NOs 7-9;
preferably, the nucleic acid sequence has a sequence as shown in any one of SEQ ID NO 10-12.
6. A nucleic acid sequence encoding an antibody to a DNA polymerase, wherein the nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence, wherein the nucleotide sequence encoding a heavy chain variable region amino acid sequence comprises a nucleotide sequence encoding a heavy chain variable region amino acid sequence set forth in any one of SEQ ID NOs 1-3, and the nucleotide sequence encoding a light chain variable region amino acid sequence comprises a nucleotide sequence encoding a light chain variable region amino acid sequence set forth in any one of SEQ ID NOs 7-9;
preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 1, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 7;
preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 2, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 8;
preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region comprises a nucleotide sequence encoding the amino acid sequence of the light chain variable region shown in SEQ ID NO. 9.
7. An expression vector comprising the nucleic acid sequence of any one of claims 4 to 6.
8. A recombinant host cell comprising the expression vector of claim 7.
9. A method for producing an antibody fragment or antibody, characterized in that the recombinant host cell according to claim 8 is used to express the antibody fragment or antibody.
10. Use of an antibody fragment according to claim 1 or 2 or an antibody according to claim 3 for selectively neutralizing the polymerization activity of a DNA polymerase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811031800.4A CN110878124B (en) | 2018-09-05 | 2018-09-05 | Antibody fragment of DNA polymerase, antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811031800.4A CN110878124B (en) | 2018-09-05 | 2018-09-05 | Antibody fragment of DNA polymerase, antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110878124A true CN110878124A (en) | 2020-03-13 |
| CN110878124B CN110878124B (en) | 2021-10-08 |
Family
ID=69727566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811031800.4A Active CN110878124B (en) | 2018-09-05 | 2018-09-05 | Antibody fragment of DNA polymerase, antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110878124B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114685671A (en) * | 2020-12-30 | 2022-07-01 | 深圳华大生命科学研究院 | Monoclonal antibody specifically combined with Taq DNA polymerase and application thereof |
| CN116731184A (en) * | 2022-11-25 | 2023-09-12 | 厦门康基生物科技有限公司 | Monoclonal antibody F6H12 specifically binding to Taq enzyme and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010082640A1 (en) * | 2009-01-15 | 2010-07-22 | 北海道三井化学株式会社 | Enzymatic preparation containing thermostable dna polymerase, process for producing same, and method for detecting analyte organism |
| CN103820415A (en) * | 2014-02-25 | 2014-05-28 | 沈鹤霄 | Preparation method of hot start DNA polymerase, prepared polymerase and application |
| CN107502600A (en) * | 2017-07-26 | 2017-12-22 | 李桂秋 | A kind of double thermal starting archaeal dna polymerases and PCR amplification detection methods containing nano antibody |
-
2018
- 2018-09-05 CN CN201811031800.4A patent/CN110878124B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010082640A1 (en) * | 2009-01-15 | 2010-07-22 | 北海道三井化学株式会社 | Enzymatic preparation containing thermostable dna polymerase, process for producing same, and method for detecting analyte organism |
| CN103820415A (en) * | 2014-02-25 | 2014-05-28 | 沈鹤霄 | Preparation method of hot start DNA polymerase, prepared polymerase and application |
| CN107502600A (en) * | 2017-07-26 | 2017-12-22 | 李桂秋 | A kind of double thermal starting archaeal dna polymerases and PCR amplification detection methods containing nano antibody |
Non-Patent Citations (3)
| Title |
|---|
| H I YAMANAKA等: "An improved phage display antibody cloning system using newly designed PCR primers optimized for Pfu DNA polymerase", 《J BIOCHEM.》 * |
| 唐笑: "耐热DNA聚合酶的定点诱变及应用于热启动PCR的纳米抗体的淘选", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 王顺祥: "《肿瘤酶学研究》", 30 June 1996, 中国科学技术出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114685671A (en) * | 2020-12-30 | 2022-07-01 | 深圳华大生命科学研究院 | Monoclonal antibody specifically combined with Taq DNA polymerase and application thereof |
| CN114685671B (en) * | 2020-12-30 | 2023-09-26 | 深圳华大生命科学研究院 | Monoclonal antibody specifically binding to Taq DNA polymerase and application thereof |
| CN116731184A (en) * | 2022-11-25 | 2023-09-12 | 厦门康基生物科技有限公司 | Monoclonal antibody F6H12 specifically binding to Taq enzyme and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110878124B (en) | 2021-10-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114787193B (en) | Anti-polymerase monoclonal antibodies and uses thereof | |
| CN109970858B (en) | CD22 single domain antibody, nucleotide sequence and kit | |
| CN114805577B (en) | Antibody for IL-17RA protein, preparation method and application thereof | |
| CN108285485B (en) | anti-PD-1 single-domain antibody and application thereof | |
| CN111116752B (en) | Immunoglobulin-binding single domain antibody, anti-avian influenza single domain antibody, bifunctional antibody and application thereof | |
| CN110003336B (en) | PD-1 single domain antibody, nucleotide sequence and kit | |
| CN112028997B (en) | anti-CEACAM 5 nano antibody | |
| KR102504884B1 (en) | Nanoantibodies capable of binding to SFTSV and applications thereof | |
| CN112521496B (en) | Monoclonal antibody specifically binding SARS-CoV-2 Spike RBD and application thereof | |
| WO2022199106A1 (en) | Chi3l1 antibody, applications thereof, nucleic acids, recombinant expression vector system, and host cell | |
| CN110878124B (en) | Antibody fragment of DNA polymerase, antibody and application thereof | |
| CN110862455B (en) | Polypeptide capable of binding CD47 and application thereof | |
| CN115819590A (en) | Preparation and application of anti-porcine erythrocyte membrane antibody | |
| CN109306008B (en) | Single-chain antibody of swine-origin anti-classical swine fever virus and preparation method thereof | |
| CN107266576B (en) | Monoclonal antibody of 1-aminocyclopropane-1-carboxylic oxidase and application thereof | |
| CN116410322A (en) | Monoclonal antibody against human PCDH7 protein and application thereof | |
| CN106957367B (en) | anti-IDH 1R132H antibody and preparation method and application thereof | |
| CN113201069B (en) | mCherry or mEOS nano antibody and preparation method and application thereof | |
| CN110003334B (en) | Polypeptide, CD19 single domain antibody, preparation method thereof, nucleotide sequence and kit | |
| CN117683122B (en) | Antibody against monkey poxvirus, and preparation method and application thereof | |
| CN110872355B (en) | Anti-amantadine AMD single-chain antibody scFv and preparation method and application thereof | |
| CN113999306B (en) | Method for obtaining antibody for recognizing space conformation epitope | |
| CN117659177B (en) | Antibodies against monkey poxvirus or antigen binding fragments thereof and uses thereof | |
| CN111349170B (en) | Monoclonal antibody of immune related GTPase family M (IRGM) and application thereof | |
| CN111349157B (en) | Monoclonal antibody of cadherin 6 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |