A hybridoma cell secreting a kOD DNA polymerase monoclonal antibody, the monoclonal antibody and application thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to a hybridoma cell secreting KOD DNA polymerase monoclonal antibody, the monoclonal antibody and application thereof. The present application utilizes the hybridoma cell to produce the monoclonal antibody capable of specifically binding with KOD DNA polymerase, and the enzyme can be released under the condition of heat activation, so that the polymerization and exonuclease functions are restored. The monoclonal antibody of the present application can be widely applied to PCR amplification, DNA sequencing, gene cloning, protein expression, protein purification and other technologies in the field of molecular biology. The monoclonal antibody can also be used in diagnosis, treatment and prevention and other aspects in the field of biomedicine, such as used for detecting pathogenic microorganisms, tumor markers, gene mutations and the like, and used for gene therapy and protein therapy and the like for treating certain diseases, such as applied to the synthesis of cDNA second strand in the mRNA sequencing library construction process in the second-generation sequencing, so as to improve the sequencing quality and accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a hybridoma cell secreting a KOD DNA polymerase monoclonal antibody, the monoclonal antibody and its application. BACKGROUND

[0002] DNA polymerase is an indispensable enzyme in biological research, widely used in PCR technology, gene cloning, gene expression and other fields. However, conventional DNA polymerase needs to be activated at high temperature, which will cause enzyme inactivation, degradation and low efficiency of polymerization reaction. Therefore, the heat-activated enzyme becomes a necessary choice in PCR technology, which can be activated at high temperature and maintain high efficiency of polymerization reaction in the early stage of PCR reaction. KOD DNA polymerase is a commonly used heat-activated enzyme, which has the advantages of high efficiency, high fidelity and high amplification capacity, and is an important choice in PCR technology. The common heat-activated modification methods on the market mainly include chemical method, ligand method and antibody method, etc. The commonly used antibody method of heat-activated DNA polymerase helps to improve the stability of reagents and solve the problem of non-specific amplification. The traditional antibody method of heat-activated enzyme is to use the polymerase activity of DNA polymerase to block the 5'~3' polymerase activity of antibody, so as to prevent non-specific amplification caused by mismatch or primer dimer at low temperature. When heated at 95℃ in PCR pre-denaturation, the antibody protein is denatured, and the polymerase activity is released, which does not affect normal amplification. The polymerase activity blocking antibody can effectively prevent non-specific amplification caused by mismatch, and improve the stability of the whole premix reaction system. However, KOD DNA polymerase also has some shortcomings, such as being easily inhibited and having weak exonuclease activity, which will affect the accuracy and efficiency of PCR reaction. Therefore, it has important theoretical and application value to develop a monoclonal antibody which can specifically bind to KOD DNA polymerase and neutralize its polymerization activity and exonuclease activity.

[0003] In addition, with the rapid development of high-throughput sequencing technology, NGS has become an important means of biological research. In NGS, the synthesis of cDNA second strand in mRNA sequencing library construction process is a very critical step, and its quality and accuracy will directly affect the reliability of subsequent sequencing results. The high efficiency and high fidelity of KOD DNA polymerase make it an excellent choice. Therefore, it has important application value to use KOD DNA polymerase for the synthesis of cDNA second strand in mRNA sequencing library construction process in NGS.

[0004] Currently, there is no commercially available B family polymerase hot start antibody, and the 5D3 and 3G8 antibodies disclosed in the literature are all non-hot start polymerase complex template amplification specific, low amplification efficiency, low detection sensitivity, and gradually eliminated by the market. Therefore, the present application provides an antibody capable of directly preparing a hot start KOD DNA polymerase, which can completely inhibit the 5'-3' nucleic acid polymerase activity and 3'-5' exonuclease activity of the KOD DNA polymerase at room temperature, and can be applied to the field of high-throughput sequencing (NGS). SUMMARY

[0005] Therefore, the present application provides a hybridoma cell secreting a KOD DNA polymerase monoclonal antibody, a monoclonal antibody and its application. The prepared antibody can specifically bind to the antigen KOD DNA polymerase, has the characteristics of high affinity, and can specifically neutralize the polymerization activity and exonuclease activity of the KOD DNA polymerase.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides an antibody fragment, which includes:

[0008] The CDR1 of the heavy chain has:

[0009] (1) the amino acid sequence shown in SEQ ID NO: 3; or

[0010] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in (1), and the function is the same or similar to that of (1); or

[0011] (3) an amino acid sequence having at least 75% homology with the amino acid sequence shown in (1) or (2);

[0012] The CDR2 of the heavy chain has:

[0013] (4) the amino acid sequence shown in SEQ ID NO: 4; or

[0014] (5) an amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in (4), and the function is the same or similar to that of (4); or

[0015] (6) an amino acid sequence having at least 75% homology with the amino acid sequence shown in (4) or (5);

[0016] The CDR3 of the heavy chain has:

[0017] (7) the amino acid sequence shown in SEQ ID NO: 5; or

[0018] (8) the amino acid sequence shown in (7) in which one or more residues are substituted, deleted or added, and which has the same or a similar function as (7); or

[0019] (9) an amino acid sequence which is at least 80% homologous to the amino acid sequence shown in (7) or (8);

[0020] the CDR1 of the light chain thereof has:

[0021] (10) the amino acid sequence shown in SEQ ID NO: 8; or

[0022] (11) the amino acid sequence shown in (10) in which one or more residues are substituted, deleted or added, and which has the same or a similar function as (10); or

[0023] (12) an amino acid sequence which is at least 60% homologous to the amino acid sequence shown in (10) or (11);

[0024] the CDR2 of the light chain thereof has an amino acid sequence comprising DTS;

[0025] the CDR3 of the light chain thereof has:

[0026] (13) the amino acid sequence shown in SEQ ID NO: 9; or

[0027] (14) the amino acid sequence shown in (13) in which one or more residues are substituted, deleted or added, and which has the same or a similar function as (13); or

[0028] (15) an amino acid sequence which is at least 67% homologous to the amino acid sequence shown in (13) or (14);

[0029] the number of the plurality is 2 to 3.

[0030] In some embodiments of the present application, the above antibody fragment has:

[0031] the heavy chain variable region thereof has:

[0032] (16) the amino acid sequence shown in SEQ ID NO: 2; or

[0033] (17) the amino acid sequence shown in (16) in which one or more residues are substituted, deleted or added, and which has the same or a similar function as (16); or

[0034] (18) an amino acid sequence which is at least 75% homologous to the amino acid sequence shown in (16) or (17);

[0035] a light chain variable region having:

[0036] (19) an amino acid sequence as shown in SEQ ID NO: 7; or

[0037] (20) an amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence as shown in (19), and having the same or similar function as (19); or

[0038] (21) an amino acid sequence having at least 75% homology with the amino acid sequence as shown in (19) or (20);

[0039] the number is 2 to 30.

[0040] The present application also provides a nucleic acid molecule having a nucleotide sequence encoding the above antibody fragment; or

[0041] having:

[0042] (22) a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 6; or

[0043] (23) a nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence as shown in (22), and having the same or similar function as (22); or

[0044] (24) a nucleotide sequence having at least 75% homology with the nucleotide sequence as shown in (22) or (23);

[0045] the number is 2 to 90.

[0046] The present application also provides an expression vector having the above nucleic acid molecule, and an acceptable genetic element.

[0047] The present application also provides a host cell having any of the following:

[0048] (i) the above antibody fragment;

[0049] (ii) the above nucleic acid molecule;

[0050] (iii) the above expression vector;

[0051] The host cell includes a hybridoma cell.

[0052] In some embodiments of the present application, the above host cell is a hybridoma cell secreting the above antibody fragment.

[0053] The application also provides application of the antibody fragment, the nucleic acid molecule, the expression vector or the host cell in preparing a hot start KOD DNA polymerase.

[0054] The application also provides a composition comprising the antibody fragment and the KOD DNA polymerase.

[0055] The application also provides a reagent or a kit comprising the antibody fragment, the nucleic acid molecule, the expression vector or the composition, and an acceptable adjuvant or auxiliary agent.

[0056] The application also provides a method for preparing the antibody fragment, comprising:

[0057] (a), introducing the nucleic acid molecule into a host cell for expression to obtain the antibody fragment; or

[0058] (b), introducing the expression vector into a host cell for expression to obtain the antibody fragment; or

[0059] (c), culturing the host cell to obtain the antibody fragment.

[0060] The application also provides a hybridoma cell secreting an anti-KOD DNA polymerase monoclonal antibody, wherein the hybridoma cell can stably secrete the KOD DNA polymerase monoclonal antibody.

[0061] In some specific embodiments of the application, the monoclonal antibody secreted by the hybridoma cell has high specificity and affinity, can specifically bind to the KOD DNA polymerase, and can release the KOD DNA polymerase under heat-activated conditions to restore the polymerization and exonuclease functions of the KOD DNA polymerase.

[0062] In some specific embodiments of the application, the monoclonal antibody can neutralize the polymerization activity and exonuclease activity of the KOD DNA polymerase.

[0063] The application also provides amino acid sequences and nucleic acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody.

[0064] The application also provides a KOD DNA polymerase monoclonal antibody fragment, wherein the antibody fragment comprises a monoclonal antibody heavy chain variable region amino acid sequence having the sequence shown in SEQ ID NO: 2.

[0065] The application also provides a KOD DNA polymerase monoclonal antibody fragment, wherein the antibody fragment comprises a monoclonal antibody light chain variable region amino acid sequence having the sequence shown in SEQ ID NO: 5.

[0066] The application further provides a nucleic acid sequence encoding a KOD DNA polymerase monoclonal antibody heavy chain variable region amino acid sequence, wherein the nucleic acid sequence comprises a nucleotide sequence encoding the heavy chain variable region amino acid sequence shown in any one of SEQ ID NO: 2-3.

[0067] Preferably, the nucleic acid sequence has the sequence shown in SEQ ID NO: 1.

[0068] The application further provides a nucleic acid sequence encoding a KOD DNA polymerase monoclonal antibody light chain variable region amino acid sequence, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding the light chain variable region amino acid sequence shown in any one of SEQ ID NO: 5-6.

[0069] Preferably, the nucleic acid sequence has the sequence shown in SEQ ID NO: 4.

[0070] The application further provides a method for secreting a KOD DNA polymerase monoclonal antibody, comprising the following steps: (1) extracting mouse B lymphocytes and myeloma cells; (2) fusing the mouse B lymphocytes and myeloma cells; (3) performing multiple screening and cloning on the fused cells to obtain hybridoma cells secreting the KOD DNA polymerase monoclonal antibody.

[0071] The application further provides a purification method for secreting a KOD DNA polymerase monoclonal antibody, comprising the following steps: (1) culturing the hybridoma cells secreting the KOD DNA polymerase monoclonal antibody; (2) separating the required monoclonal antibody by using a centrifugation method or an ultrafiltration method; and (3) purifying and identifying by using an enzyme-linked immunosorbent method or the like.

[0072] The application further provides a preparation method of a hot-start enzyme reagent, comprising the following steps: (1) mixing KOD DNA polymerase monoclonal antibodies and KOD DNA polymerase according to a certain ratio; and (2) incubating at 25 DEG C for 1 hour.

[0073] The application further provides a monoclonal antibody applied to construction of a second-generation sequencing library, which can block KOD DNA polymerase activity at room temperature, and can release the enzyme under the condition of heat activation, so that the enzyme restores polymerization and exonuclease functions.

[0074] The application has the following effects:

[0075] The hybridoma cell and the monoclonal antibody secreted therefrom have the effect of reducing non-specific amplification. The monoclonal antibody secreted by the hybridoma cell can completely block the activity of DNA polymerase, and the heat-start enzyme after blocking can restore the activity of DNA polymerase after heating at high temperature for 30s-10min. BRIEF DESCRIPTION OF DRAWINGS

[0076] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced as follows.

[0077] Figure 1 SDS-PAGE diagram of the purified 24# monoclonal antibody is shown, wherein the left lane shows non-reduction, and the right lane shows reduction;

[0078] Figure 2 HPLC diagram of the purified 24# monoclonal antibody is shown;

[0079] Figure 3 PCR diagram of 24# antibody nucleic acid residue detection is shown;

[0080] Figure 4 Electrophoresis diagram of 24# antibody nuclease residue activity detection is shown;

[0081] Figure 5 DNA urea PAGE electrophoresis detection result of antibody blocking KOD DNA polymerase polymerization activity is shown;

[0082] Figure 6 Antibody heat-start KOD DNA polymerase PCR performance evaluation (different GC amplification results) is shown;

[0083] Figure 7 Antibody heat-start KOD DNA polymerase PCR performance evaluation (four-way PCR amplification results) is shown. DETAILED DESCRIPTION

[0084] The present application discloses a hybridoma cell secreting KOD DNA polymerase monoclonal antibody, a monoclonal antibody and its application, and those skilled in the art can refer to the content of the present application to appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0085] The present application relates to the technical field of hot start enzyme antibody preparation, and particularly relates to a hybridoma cell specifically binding to KOD DNA polymerase, a monoclonal antibody and application thereof, which are applied to PCR amplification, DNA sequencing, gene cloning, protein expression, protein purification and the like in the technical field of molecular biology. The monoclonal antibody can also be used in diagnosis, treatment and prevention and the like in the biomedical field, such as detection of pathogenic microorganisms, tumor markers, gene mutations and the like, and gene therapy and protein therapy for treating certain diseases, such as application to synthesis of cDNA second strand in the mRNA sequencing library construction process in the second generation sequencing, so as to improve sequencing quality and accuracy.

[0086] The hybridoma cell of the present application is obtained by fusing mouse B lymphocytes with myeloma cells and is obtained through multiple screening and cloning. The hybridoma cell can stably secrete the KOD DNA polymerase monoclonal antibody. The monoclonal antibody has high specificity and affinity and can specifically bind to the KOD DNA polymerase, so as to play a role in blocking enzyme activity in the PCR amplification reaction.

[0087] The present application provides a preparation method of the KOD DNA polymerase monoclonal antibody, which comprises the following steps: emulsifying the antigen KOD DNA polymerase with an equal volume of adjuvant, immunizing a mouse for 3 times, collecting spleen cells from the spleen, and hybridizing and fusing the spleen cells with myeloma cells; obtaining the monoclonal antibody hybridoma cell through 3 rounds of screening by using the traditional hybridoma technology; injecting the monoclonal antibody hybridoma cell strain into the abdominal cavity of a Balb / c mouse, collecting the ascites after 7 days; and purifying the ascites by the caprylic acid-ammonium sulfate method to obtain the monoclonal antibody.

[0088] The present application provides a monoclonal antibody, which is secreted by the hybridoma cell and can specifically bind to the KOD DNA polymerase. The monoclonal antibody provided by the present application has the following characteristics: 1) specifically blocking the KOD DNA polymerase; 2) high purity; 3) no nucleic acid and nuclease residues in the antibody; 4) good blocking effect on polymerization and exonuclease activity of the KOD DNA polymerase; and 5) the KOD DNA polymerase is used for synthesis of cDNA second strand in the mRNA sequencing library construction process in the NGS.

[0089] The monoclonal antibody of the present application can be widely applied to PCR amplification, DNA sequencing, gene cloning, protein expression, protein purification and the like in the technical field of molecular biology. The monoclonal antibody can also be used in diagnosis, treatment and prevention and the like in the biomedical field, such as detection of pathogenic microorganisms, tumor markers, gene mutations and the like, and gene therapy and protein therapy for treating certain diseases.

[0090] The monoclonal antibody of the present application can also be widely applied in the field of high-throughput sequencing (NGS). In NGS, PCR amplification is one of the key steps for establishing a sequencing library. Due to various factors in the PCR amplification process that can cause amplification bias and errors, efficient, specific and stable PCR enzymes are needed for amplification reactions. The monoclonal antibody of the present application has high specificity and affinity, and can specifically bind to KOD DNA polymerase, thereby playing a highly efficient hot start role in PCR amplification reactions. Therefore, the monoclonal antibody of the present application can be used as an important component of PCR amplification reactions in the establishment of NGS sequencing libraries, improve the specificity and stability of PCR amplification reactions, and thus improve the accuracy and reliability of NGS sequencing.

[0091] The present application also provides a method for preparing a hot start enzyme reagent, comprising the following steps: co-incubating a monoclonal antibody with KOD DNA polymerase, and the mixing ratio of the monoclonal antibody and the DNA polymerase affects the blocking effect of the monoclonal antibody on the DNA polymerase.

[0092] The sequence information related to the present application is as follows:

[0093] The nucleic acid sequence of the heavy chain variable region of monoclonal antibody 24# is as follows:

[0094] GAGGTGAAGCTGGTGGAGTCTGGGGGAGACTTATTGGAGCCTGGAGGGTCCCTAAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCTTCTATCCAGACAATATGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAGGACGATATAGGTACGACGTGAGATACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 1)

[0095] The amino acid sequence of the heavy chain variable region of monoclonal antibody 24# is as follows:

[0096] EVKLVESGGDLLEPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSGGSYTFYPDNMKGRFTISRD NAKNTLYLQMSSLKSEDTAMYYCAGRYRYDVRYYAMDYWGQGTSVTVSS (SEQ ID NO: 2)

[0097] Amino acid sequence of the CDR1, CDR2, CDR3 regions of the heavy chain of monoclonal antibody 24#:

[0098] GFTFSSYG (SEQ ID NO: 3).... ISSGGSYT (SEQ ID NO: 4).. AGRYRYDVRYYAMDY (SEQ ID NO: 5)

[0099] Nucleic acid sequence of the variable region of the light chain of monoclonal antibody 24#:

[0100] GACATTGTTCTCACCCAGTCTCCAGCAATCATGTCGGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTGAGTTACTTGTATTGGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTGGTTACCCACCCAAGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG (SEQ ID NO: 6)

[0101] Amino acid sequence of the variable region of the light chain of monoclonal antibody 24#:

[0102] DIVLTQSPAIMSASPGEKVTMTCSASSSVSYLYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLT ISRMEAEDAATYYCQQWSGYPPKYTFGGGTKLEIKR (SEQ ID NO: 7)

[0103] Amino acid sequence of the CDR1, CDR2, CDR3 regions of the light chain of monoclonal antibody 24#:

[0104] SSVSY (SEQ ID NO: 8)..................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................... DTS..................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................... QQWSGYPPKYT (SEQ ID NO: 9)

[0105] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present application are ordinary commercially available products, which can be purchased from the market.

[0106] As used herein, "comprise", "include", "have", "contain", and the like, are open-ended terms, i.e., meaning "including but not limited to".

[0107] The antibody fragment described in the present application also refers to a complete antibody.

[0108] The present application is further described below in conjunction with examples:

[0109] Example 1: Preparation of anti-KOD DNA polymerase monoclonal antibody

[0110] 1. Mouse immunization

[0111] The KOD DNA polymerase protein (KOD DNA polymerase, Zheyebio, ZY101002) was emulsified with Freund's complete adjuvant, and then 5-week-old female Balb / c mice were immunized intraperitoneally. The initial dose was 100 μg per mouse. The second and third immunizations were performed at intervals of 21 days and 42 days after the first immunization, respectively, and the immunization dose was 50 μg per mouse. About 10 days after the third immunization, blood was collected from the tail, and the serum titer was detected by indirect method using a 96-well plate coated with KOD DNA polymerase. The results are shown in Table 1.

[0112] Table 1: Detection of KOD DNA polymerase antibody mouse serum subtypes

[0113] Mouse No. 1 / 200 1 / 1K 1 / 2K 1 / 4K 1 / 8K 1 / 16K 1 / 32K 1 / 64K 1 / 128K 1 / 256K 1 / 512K Control 1# 2.909 2.753 1.748 1.928 1.532 1.531 1.098 1.048 0.41 0.366 0.191 0.04 2# 2.798 3.014 3.408 3.058 2.503 2.294 1.674 1.084 0.621 0.364 0.202 0.045 3# 3.444 3.344 2.745 2.731 1.802 1.328 0.752 0.538 0.283 0.19 0.127 0.051 4# 2.505 2.731 2.516 1.978 1.617 1.085 0.713 0.457 0.258 0.181 0.112 0.052 5# 2.806 2.734 2.537 2.06 2.053 1.266 0.902 0.578 0.349 0.19 0.131 0.051 6# 2.824 2.568 2.114 1.451 1.2 0.637 0.46 0.268 0.174 0.112 0.076 0.05 7# 2.583 2.723 2.654 2.446 2.134 1.454 1.189 0.596 0.364 0.237 0.149 0.048 8# 2.357 2.541 2.486 1.931 1.533 0.945 0.661 0.359 0.234 0.139 0.092 1.779 B 0.077 0.054 0.046 0.042 0.042 0.051 0.046 0.056 0.042 0.044 0.044 0.044

[0114] 2. Hybridoma cell preparation

[0115] Mice with serum titer greater than 10 4 were selected for intrasplenic booster immunization at a dose of 100 μg per mouse. Three days after booster immunization, the mouse spleen was removed and fused with mouse myeloma cells NS1 at a ratio of 10:1. The fused cells were cultured in DMEM medium (Gibco) containing HAT.

[0116] About 6-7 days after fusion, the specific antibody content in the cell culture supernatant was detected by indirect method using a 96-well plate coated with KOD DNA polymerase. Positive wells with OD value not less than 0.5 were selected for 3 rounds of subcloning by limiting dilution method, and finally hybridoma cell strains stably secreting anti-KOD DNA polymerase were obtained.

[0117] 3. Purification of monoclonal antibody against KOD DNA polymerase

[0118] The obtained mouse hybridoma cells stably secreting the antibody against KOD DNA polymerase were injected into the abdominal cavity of a mouse, and ascites was collected to obtain a monoclonal antibody against KOD DNA polymerase with a purity of more than 90% by high-throughput antibody purification, removing other unbound antibodies and nucleic acids and nucleases and other impurities. The purification experiment results of the 24# antibody are shown in detail in Table 1. Figure 1 to Figure 4 .

[0119] 4. Subtype identification of KOD DNA polymerase monoclonal antibody

[0120] KOD DNA polymerase was diluted to 1 μg / ml and coated on a 96-well enzyme-free plate overnight, and then blocked with 1% Casein. The purified KOD DNA polymerase monoclonal antibody (concentration of 5 mg / ml) was diluted with 0.05 mmol / L CB buffer at pH 9.6 at a ratio of 1:1000. The subtype of the purified KOD DNA polymerase antibody was identified by a mouse monoclonal antibody subtype identification reagent indirect method, and the results are shown in Table 2.

[0121] Table 2: Subtype identification results of KOD DNA polymerase antibody

[0122] IgGl IgG2a IgG2b IgG3 IgM IgA 1# 0.674 0.041 0.041 0.044 0.051 0.047 2# 0.582 0.044 0.044 0.051 0.051 0.043 3# 0.551 0.052 0.045 0.044 0.049 0.05 4# 0.471 0.066 0.061 0.069 0.055 0.045 5# 0.049 0.049 0.33 0.056 0.069 0.049 6# 0.515 0.059 0.059 0.07 0.052 0.045 7# 0.724 0.046 0.049 0.062 0.06 0.045 8# 0.533 0.047 0.046 0.049 0.047 0.043 9# 0.708 0.097 0.045 0.043 0.053 0.06 10# 0.504 0.044 0.043 0.043 0.043 0.044 11# 0.638 0.069 0.066 0.071 0.05 0.068 12# 0.485 0.088 0.047 0.058 0.049 0.043 13# 0.495 0.054 0.045 0.046 0.048 0.061 14# 0.459 0.071 0.046 0.058 0.048 0.048 15# 0.584 0.04 0.056 0.042 0.042 0.044 16# 0.661 0.054 0.042 0.043 0.045 0.046 17# 0.611 0.044 0.044 0.045 0.052 0.053 18# 0.494 0.047 0.047 0.049 0.046 0.044 19# 0.638 0.044 0.044 0.046 0.049 0.054 20# 0.825 0.048 0.051 0.058 0.048 0.047 21# 0.794 0.045 0.047 0.042 0.046 0.045 22# 0.96 0.045 0.046 0.045 0.045 0.043 23# 0.889 0.044 0.042 0.045 0.042 0.043 24# 0.794 0.043 0.044 0.045 0.049 0.043 25# 0.983 0.047 0.053 0.052 0.059 0.051 26# 1.175 0.045 0.049 0.046 0.048 0.047 27# 1.274 0.055 0.053 0.057 0.086 0.054 28# 1.011 0.055 0.051 0.058 0.053 0.049 29# 0.973 0.048 0.047 0.051 0.051 0.046 30# 1.218 0.053 0.056 0.055 0.054 0.059 31# 1.008 0.053 0.055 0.059 0.051 0.05 32# 0.995 0.046 0.049 0.047 0.066 0.046 33# 0.976 0.049 0.049 0.048 0.076 0.052 34# 1 0.05 0.05 0.05 0.051 0.051 35# 0.977 0.05 0.058 0.053 0.053 0.053 36# 1.212 0.045 0.047 0.044 0.047 0.047 37# 0.603 0.049 0.047 0.044 0.049 0.046 38# 0.755 0.058 0.051 0.048 0.048 0.044

[0123] As can be seen from Table 2, the subtype of the KOD DNA polymerase monoclonal antibody 1 strain is IgG2b, and the subtypes of the remaining strains are IgG1.

[0124] 5. Titer detection of KOD DNA polymerase monoclonal antibody

[0125] DNA polymerase was diluted to 1 μg / ml with 0.2M pH=7.0 PB buffer, 50 μl was added to each well of 96-well enzyme-free plate (Corning) for coating overnight at 4°C. The next day, wash 3 times with PBST, then block with 1% Casein, 100 μl / well, block for 2 hours at 37°C. The purified 38 monoclonal antibodies (5 mg / ml each) were diluted by 0.2M pH=7.0 PB buffer according to the ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, …. The diluted antibodies were added to the enzyme-free plate coated with DNA polymerase, and 0.05 mmol / L CB buffer, pH=9.6 was added to the negative control wells, 50 μl / well. After 30 minutes of reaction at 37°C, wash the plate 5 times with PBST, dry, then add 1:4000 diluted HRP-goat anti-mouse IgG (SIGMA), 100 μl / well, react for 30 minutes at 37°C. Wash the plate 5 times with PBST, dry, then add the general enzyme-free substrate, 100 μl / well, react for 10 minutes at room temperature in the dark, add 50 μl of 0.1 mol / L sulfuric acid to terminate the reaction, and measure the absorbance at 450 nm. The detection results are shown in Table 3 below.

[0126] Table 3: Absorbance of KOD DNA polymerase antibody at different dilution ratios

[0127]

[0128]

[0129] The titers of various antibodies can be seen from Table 3, and antibodies with high OD values were selected for functional detection.

[0130] Example 2: Preparation of KOD DNA hot start enzyme reagent

[0131] KOD DNA polymerase (Zeyebio, ZY101002) and the antibody prepared in Example 1 were mixed in a certain ratio, the final concentration of the enzyme was 12.5 μg / ml (the molar ratio of antibody to antigen was 2:1, and the mass ratio was 30:7), and incubated at 25°C for 1 hour to obtain KOD DNA hot start enzyme.

[0132] Example 3: KOD DNA polymerase antibody blocking KOD DNA polymerase polymerization activity experiment

[0133] 1. DNA urea PAGE electrophoresis method for detecting blocking efficiency

[0134] The DNA urea PAGE electrophoresis method was used to determine whether the polymerization activity was blocked by the proportion of product bands. The polymerization activity test reaction system is shown in Table 4:

[0135] Table 4

[0136] Ingredient Volume (μL) 50 μM annealed primer 1 2x KOD Buffer 12.5 Water 10.5 KOD DNA Hot Start Enzyme (0.145 mg / mL) 1

[0137] The primer is a single-stranded primer, provided with a complementary sequence, and the product formed after annealing is not flush at the end, which is both a primer and a template. The DNA hot-start polymerase can continue to extend at the 3' end until the end is flush. Since the molecular weight of the extended DNA molecule will increase, the blocking effect of the antibody on KOD DNA polymerase is evaluated accordingly.

[0138] The KOD DNA hot-start enzyme includes the KOD DNA hot-start enzyme prepared in Example 2 or the unblocked KOD DNA hot-start enzyme.

[0139] Reaction conditions: 50°C / 30min, 30°C / 40min, then add 2*loading to terminate the reaction. Detection is performed by nucleic acid PAGE. The experimental results are shown in Table 4. Figure 5

[0140] It can be seen that through the two reaction conditions, the antibodies with better blocking of polymerization activity are 24# and 33#.

[0141] Example 4: Blocking of KOD DNA polymerase exonuclease activity by KOD DNA polymerase antibody

[0142] The conversion rate before and after adding the enzyme is used to judge the blocking efficiency of the exonuclease activity.

[0143] The exonuclease test reaction system is shown in Table 5:

[0144] Table 5

[0145] Ingredient Volume (μL) 2x KOD Buffer 12.5 10 μM annealed primer 1 Water 10.5 KOD DNA Hot Start Enzyme (0.145 mg / mL) 1

[0146] The primer is a single-stranded primer, provided with a complementary sequence, and the product formed after annealing is not flush at the end, which is both a primer and a template. The DNA hot-start polymerase can continue to extend at the 3' end until the end is flush. Since the molecular weight of the extended DNA molecule will increase, the blocking effect of the antibody on KOD DNA polymerase is evaluated accordingly.

[0147] The KOD DNA hot-start enzyme includes the benchmark KOD DNA hot-start enzyme (denoted as "benchmark"; Toyo Spinning, product number: KME-101), the KOD DNA hot-start enzyme prepared in Example 2 (6#, 11#, 13#, 15#) or the unblocked KOD DNA hot-start enzyme (denoted as "positive").

[0148] ​​qPCR program name: 37℃-30min-MB; reaction conditions: 37℃, 30min. Results are shown in Tables 6-8.

[0149] Table 6: Results of KOD DNA Polymerase Antibody Blocking and KOD DNA Polymerase Exonuclease Activity Detection

[0150] Exo-activity blocking evaluation Conversion rate Blocking efficiency 6# 165％ -20％ 11# 60％ 56％ 13# 107％ 22％ 15# 73％ 47％ Mark 10.40％ 92％ Positive 137.50％ 0％

[0151] Table 7

[0152]

[0153]

[0154] Table 8

[0155] Exo-activity blocking evaluation Conversion rate Blocking efficiency 31# 755％ -87％ 33# 274％ 32％ 34# 432％ -7％ 35# 388％ 4％ Mark 47％ 88％ Positive 404％ 0％

[0156] Conversion rate = (Final cycle fluorescence value - First cycle fluorescence value) / First cycle fluorescence value

[0157] Encapsulation efficiency = 1 - Conversion rate / Positive conversion rate

[0158] Based on the data, antibody #24 showed the best excision blocking efficiency.

[0159] Example 5: Functional test of KOD DNA hot-start enzyme reagent

[0160] 1. Validation using different GC amplification methods; reaction systems are shown in Table 9, and conditions are shown in Table 10.

[0161] Table 9

[0162] Volume (μL) 2* Buffer 10 Template 0.4 KOD DNA Hot Start Enzyme 0.4 Water 8 Primer 1.2

[0163] The KOD DNA hot-start enzyme includes a standard KOD DNA hot-start enzyme or one blocked with antibody #24.

[0164] KOD DNA hot-start enzyme. Table 10

[0165]

[0166] Different GC amplification assays were performed using a benchmark hot-start enzyme. The results are shown in [Figure number missing]. Figure 6 The amplification effect of the self-produced 24# antibody hot-start enzyme under different GC conditions is basically the same as that of the benchmark hot-start enzyme.

[0167] 2. Quadruple PCR amplification verification; the reaction system is shown in Table 11:

[0168] Table 11

[0169]

[0170]

[0171] The primer addition amount is shown in Table 12:

[0172] Table 12

[0173] Concentration Volume (μL) Primer F 10 μM 0.6 Primer R 10 μM 0.6

[0174] The primer DC-1 / 3 / 5 / 6, the extension time is 3 min.

[0175] The fourfold PCR amplification detection is carried out on the control benchmark hot start enzyme, and the experimental results are shown in Figure 7 The fourfold amplification effect of the self-produced 24# antibody hot start enzyme is basically consistent with that of the benchmark hot start enzyme.

[0176] In summary, the test results of the self-produced antibody different GC and fourfold PCR are consistent with the benchmark.

[0177] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An antibody fragment against KOD DNA polymerase, characterized in that: The amino acid sequence of CDR1 in the heavy chain is shown in SEQ ID NO: 3; The amino acid sequence of CDR2 in the heavy chain is shown in SEQ ID NO: 4; The amino acid sequence of CDR3 in the heavy chain is shown in SEQ ID NO: 5; The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 8; The amino acid sequence of the light chain CDR2 is DTS; The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

9.

2. The antibody fragment as described in claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

7.

3. A nucleic acid molecule, characterized in that, Having a nucleotide sequence encoding an antibody fragment as described in claim 1 or 2; or It has nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO:

6.

4. An expression carrier, characterized in that, It has the nucleic acid molecule as described in claim 3, and acceptable gene elements.

5. A host cell, characterized in that, It has any of the following: (i) The antibody fragment as described in claim 1 or 2; (ii) The nucleic acid molecule as described in claim 3; (iii) The expression vector as described in claim 4; The host cells include hybridoma cells.

6. The host cell as described in claim 5, characterized in that, It is a hybridoma cell that secretes the antibody fragment as described in claim 1 or 2.

7. The use of the antibody fragment as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the expression vector as described in claim 4, or the host cell as described in claim 5 or 6 in the preparation of hot-start KOD DNA polymerase.

8. A composition, characterized in that, It includes the antibody fragment as described in claim 1 or 2 and KOD DNA polymerase.

9. A reagent or kit, characterized in that, It includes the antibody fragment as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the expression vector as described in claim 4, or the composition as described in claim 8, and acceptable excipients or adjuvants.

10. A method for preparing antibody fragments against KOD DNA polymerase, characterized in that, include: (a) Introduce the nucleic acid molecule as described in claim 3 into a host cell for expression to obtain an antibody fragment; or (b) Introduce the expression vector as described in claim 4 into host cells for expression to obtain antibody fragments; or (c) Culture the host cells as described in claim 5 or 6 to obtain antibody fragments.

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