CN115594768B

CN115594768B - Hybridoma cell secreting anti-DNA polymerase monoclonal antibody, monoclonal antibody and application of monoclonal antibody

Info

Publication number: CN115594768B
Application number: CN202211587528.4A
Authority: CN
Inventors: 杨心意; 孟凤丽; 覃雨棠; 王丹霞; 翁艺恒; 时彦祎
Original assignee: Zhuhai Baorui Biotechnology Co ltd
Current assignee: Zhuhai Baorui Biotechnology Co ltd
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-03-17
Anticipated expiration: 2042-12-12
Also published as: CN115594768A

Abstract

The invention discloses a hybridoma cell secreting an anti-DNA polymerase monoclonal antibody, a monoclonal antibody and application thereof, and relates to the technical field of preparation of hot-start enzyme antibodies. The monoclonal antibody secreted by the hybridoma cell provided by the invention can completely block the activity of DNA polymerase. Compared with the existing hot-start enzyme, the DNA polymerase modified by the monoclonal antibody has better stability, and the normal-temperature storage time of the hot-start enzyme is obviously prolonged. The monoclonal antibody secreted by the hybridoma cells has no nuclease activity residue. In addition, the monoclonal antibody obtained by screening can keep good low-temperature blocking effect and high-temperature release polymerization activity effect on various DNA polymerases, and has good universality. The monoclonal antibody provided by the invention has the advantages of small dosage and good blocking activity.

Description

Hybridoma cell secreting anti-DNA polymerase monoclonal antibody, monoclonal antibody and application of monoclonal antibody

Technical Field

The invention relates to the technical field of preparation of hot-start enzyme antibodies, and particularly relates to a hybridoma cell secreting an anti-DNA polymerase monoclonal antibody, the monoclonal antibody and application of the monoclonal antibody.

Background

Polymerase Chain Reaction (PCR) technology is widely applied to molecular biology experiments, and can complete 10 DNA molecules of 1 DNA molecule in vitro in a short time (several hours) ⁹ And (5) performing amplification. The PCR technology is similar to the natural replication process of double-stranded DNA, namely, the process of in vitro replication and synthesis of daughter strand DNA complementary to the mother strand template DNA by taking the mother strand DNA as a template and a specific primer as an extension origin under the catalytic action of DNA polymerase through the steps of pre-denaturation, annealing, extension and the like. While DNA polymerases are the key to achieving DNA replication.

When DNA replication, it is known that nonspecific strand extension by DNA polymerase generally occurs before the start of thermal cycling, all reactants are mixed at room temperature, resulting in the appearance of nonspecific amplification products. The emergence of the "hot start" enzyme, in turn, significantly reduced non-specific amplification during DNA replication. The common feature of hot-start enzymes is their thermostability, at lower temperatures the activity of the enzyme is inhibited and at higher temperatures matched to the PCR reaction, e.g.hot-start DNA polymerases.

Tth DNA polymerase, one of the DNA polymerases, is derived from thermophilic eubacteriumThermus thermophilusHB8 has been isolated, has thermal stability and reverse transcriptase activity, and is a DNA polymerase commonly used in PCR technology. This enzyme is a highly processive 5'-3' DNA polymerase lacking 3'-5' exonuclease activity (proofreading). Its intrinsic Reverse Transcriptase (RT) activity is in Mn ²⁺ Under the existing conditions, the transcription efficiency is far higher than that of Taq DNA polymerase, and the DNA polymerase is more tolerant to inhibitory components such as blood-borne inhibitors. Therefore, tth DNA polymerase can be widely used in various specific PCR reactions.

However, most of DNA polymerases including Tth DNA polymerase still have a certain polymerase activity at low temperature, which results in non-specificity and primer dimer amplification during PCR amplification, and long-term stability of the enzyme.

With the wide application of PCR technology and the gradual improvement of the requirement on PCR amplification quality, the appearance of hot-start enzyme technology can improve the enzymological properties of common DNA polymerase, and the current common methods comprise antibody modification, ligand modification, chemical modification and the like, wherein the use of the hot-start enzyme modified by the antibody is more common.

The antibody modified DNA polymerase needs to use a specific anti-DNA polymerase monoclonal antibody, the specific anti-DNA polymerase monoclonal antibody is combined with the DNA polymerase to form an antigen-antibody complex, and the activity of the DNA polymerase can be effectively blocked at room temperature, so that the DNA polymerase does not exert polymerase activity at low temperature; at high temperature, the complex dissociates to release active DNA polymerase, which then performs PCR amplification reaction, thus effectively avoiding primer dimer formation, reducing non-specific product amplification, and extending the long-term stability of DNA polymerase. However, only a few anti-Taq enzyme monoclonal antibodies, such as a Hieff anti-Taq DNA Polymerase enzyme monoclonal Antibody and a Hieff anti-exouclase of Taq anti-Taq enzyme monoclonal Antibody 5'→ 3' exonuclease monoclonal Antibody, are available on the market, and there are few monoclonal antibodies having blocking activity against a variety of DNA polymerases. Although patent CN202010994643.8 provides a Tth DNA polymerase monoclonal antibody, it cannot achieve 100% effect of blocking Tth DNA polymerase, and the existing hot start Tth DNA polymerase has poor stability.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a hybridoma cell and a monoclonal antibody secreting a DNA polymerase-resistant monoclonal antibody, wherein the monoclonal antibody secreted by the hybridoma cell has good blocking activity on various DNA polymerases, and particularly can achieve a 100% blocking effect on Tth DNA polymerase, so that the prepared hot-start enzyme has good stability, can still maintain good blocking effect and amplification effect after being placed for more than 15 days at normal temperature, is beneficial to reducing the requirement of an amplification detection reagent or a kit on temperature (low temperature) during transportation and storage, and widens the application scene of the hot-start enzyme.

The invention is realized by the following steps:

in a first aspect, the present invention provides a hybridoma cell secreting a monoclonal antibody against DNA polymerase, which is deposited at the Guangdong province culture Collection of microorganisms at the following locations: the strain preservation center of the institute of microorganism research on 5 th floor of experimental building of 100 # university in the overseas district of Guangzhou City, guangdong province has the preservation numbers: GDMCC:62992 with a storage date of: 22/11/2022, deposited under the cell name: hybridoma cell TtA, identified as: survival, taxonomic name: hybridoma cell lines.

The antibody secreted by the hybridoma cell has the activity of completely blocking DNA polymerase at low temperature, so that the DNA polymerase does not exert polymerase activity at low temperature. At high temperatures, the monoclonal antibody is detached from the binding site of the DNA polymerase, releasing the activity of the DNA polymerase. Then PCR amplification reaction is carried out, thus effectively avoiding the formation of primer dimer and reducing the amplification of non-specific products. Therefore, the monoclonal antibody secreted by the hybridoma cell provided by the invention has the following advantages: 1. has the effect of reducing amplification of non-specific reaction. 2. The monoclonal antibody can completely block the activity of DNA polymerase, and the activity of the DNA polymerase can be recovered by heating the blocked hot-start enzyme at high temperature for 30s-10 min. 3. Compared with the existing hot-start enzyme, the DNA polymerase modified by the monoclonal antibody has better stability (especially thermal stability), and the normal-temperature storage time of the hot-start enzyme is obviously prolonged. 4. The monoclonal antibody has no residual DNA lyase activity, no residual exonuclease activity and no residual endonuclease activity. 5. The monoclonal antibody secreted by the hybridoma cell can keep good low-temperature blocking effect and high-temperature release polymerization activity effect on various DNA polymerases, and has universality.

In a second aspect, the present invention also provides a method for preparing a hybridoma cell, comprising the steps of:

immunizing an animal capable of generating a monoclonal antibody by using DNA polymerase, detecting the titer of animal serum and the blocking and/or starting activity of the animal serum on the activity of the DNA polymerase, and screening to obtain an animal capable of being used for cell fusion;

fusing the screened spleen cells of the animals with myeloma cells to obtain hybridoma cells;

hybridoma cells were screened and cloned.

Selecting serum with high animal serum titer, and eliminating animals without closed DNA polymerase activity by primarily screening the influence of the animal serum on the closed activity of the DNA polymerase activity and the DNA polymerase starting activity so as to improve the probability of screening hybridoma cells with high closed DNA polymerase activity and even completely closed DNA polymerase activity.

In an alternative embodiment, the cell fusion is performed with animals having a serum titer above 1 10000 and a low serum dose that completely blocks DNA polymerase activity.

Screening to obtain animals capable of being used for cell fusion, wherein one screening method comprises the following steps:

selecting animals of which the dosage a of the animal serum has obvious DNA polymerase blocking effect and can completely block the DNA polymerase activity under the condition of the animal serum dosage higher than the dosage a; the dosage of a is 0.5-1 μ l of animal serum stock solution.

In an alternative embodiment, the above amount of more than a refers to 1 < a <10 μ l of animal serum stock. For example 1 < a < 4. Mu.l, or 2 < a < 4. Mu.l.

In other embodiments, animals that completely block DNA polymerase activity at a dose of 1 μ l animal serum when 0.5 μ l animal serum stock has significant DNA polymerase blocking effect are also within the scope of the invention.

The other screening method comprises the following steps: selecting animals of which the dosage b can completely block the activity of the DNA polymerase; the dosage of b refers to 0.5-2 μ l of animal serum stock solution.

For example, screening 0.5-1 μ l of animal serum stock solution can completely block DNA polymerase activity; or screening 1-2 μ l animal serum stock solution to completely block DNA polymerase activity.

In one embodiment, b =2a may also be provided. For example, the amount of a is 0.5-1. Mu.l of the stock animal serum, and the amount of b is 1-2. Mu.l of the stock animal serum.

Compared with a single ELISA detection method, the screening strategy greatly improves the accuracy of target animals, thereby increasing the possibility of screening the monoclonal antibody capable of completely blocking the Tth DNA polymerase activity.

The cell fusion method includes, but is not limited to, the PEG method, sendai virus method, receptor targeting method, electrofusion method, and magnetic field fusion method.

Screening methods for hybridoma cells include, but are not limited to: solid phase radioimmunoassay, solid phase enzyme immunoassay, immunofluorescence, indirect hemagglutination assay, microcytotoxicity assay or spot assay. So long as hybridoma cells capable of secreting monoclonal antibodies reactive with DNA polymerase can be screened.

Cloning of hybridoma cells includes, but is not limited to: limiting dilution, micromanipulation, soft agar plate, and cell sorter.

For example, the cloning of hybridoma cells is carried out by a limiting dilution method which does not require any special equipment and is high in the efficiency of the occurrence of cloned cells. The cell suspension was serially diluted so that each culture well contained a specific number of cells.

In a preferred embodiment of the present invention, the cloning of the hybridoma cells comprises: positive hybridoma cells were subjected to three monoclonality. Three monoclonals were performed to ensure that each well was monoclonal.

In an alternative embodiment, during cloning, 1 96 well cell culture plate is plated per single cloning, and during the first single cloning, 5 cells/well are placed; second monoclonalization: set 3 cells/well; third monoclonality: set 1 cell/well.

Each clone was tested according to cell clone size and OD _450nm The detection values are comprehensively evaluated, and the monoclonal cells with moderate cell clone size and high positive value are selected for plating so as to avoid long-term competitive growth among negative hybridoma cells, non-monoclonal cells with weak antibody secreting ability or low affinity antibody secreting ability, and ensure effective growth and stability of the monoclonal cells with strong antibody secreting ability and high affinity antibody secreting ability.

In an alternative embodiment, the animal capable of producing monoclonal antibodies is a mouse, rat or rabbit.

In a third aspect, the present invention provides a monoclonal antibody secreted by the hybridoma cell described above, the monoclonal antibody being capable of specifically binding to a DNA polymerase.

The monoclonal antibody provided by the invention has the following characteristics by identification: 1) The purity is high; 2) Residual activity after DNA polymerase blocking was low: 3) The blocking activity is good, the experiment requirement can be met with low dosage, and compared with the blocking dosage of 1ug monoclonal antibody/5U DNA polymerase in the prior art, the monoclonal antibody provided by the invention has the advantages of low dosage and good blocking activity; 4) The DNA polymerase modified by the antibody has good starting activity; 5) No nuclease residue, no DNA lyase activity residue, no exonuclease activity residue and no endonuclease activity residue.

The subtype of the monoclonal antibody is IgG1.

In a fourth aspect, the present invention also provides a method for preparing a monoclonal antibody, which comprises the following steps: the hybridoma cell or the hybridoma cell prepared by the preparation method of the hybridoma cell is inoculated into an animal, and ascites is collected.

In a preferred embodiment of the present invention, the ascites fluid is collected and then subjected to a blocking and/or priming activity test of DNA polymerase, and the ascites fluid is screened for an activity capable of completely blocking the DNA polymerase.

The ascites fluid capable of completely blocking the activity of the DNA polymerase is screened by previously performing the blocking and/or priming activity test of the DNA polymerase on the ascites fluid of the inoculated animal, and then the ascites fluid capable of completely blocking the activity of the DNA polymerase is subjected to antibody minification. The low activity of the ascites blocking DNA polymerase obtained by screening is avoided.

In an alternative embodiment, the collection of ascites fluid further comprises subtyping and antibody purification of the ascites fluid.

In an alternative embodiment, the antibody purification is performed using at least one of affinity chromatography, gel filtration chromatography, ion exchange chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography, and ammonium sulfate precipitation.

The packing material of the chromatographic column of the affinity chromatography is at least one of Protein A, protein G or Protein L.

In a fifth aspect, the invention also provides a nucleic acid molecule encoding the monoclonal antibody described above.

In a sixth aspect, the present invention also provides a recombinant vector comprising the nucleic acid molecule described above.

In a seventh aspect, the present invention also provides a hot start enzyme, which comprises a DNA polymerase modified by an antibody, wherein the antibody is the monoclonal antibody described above, and the monoclonal antibody is bound to the DNA polymerase through an antigen-antibody reaction.

The hot-start enzyme can still keep a stable sealing effect after being heated for 1h at the temperature of 30-60 ℃, can meet the experiment requirements under most of the existing experiment conditions, and has a good application prospect. After the amplification system containing the hot-start enzyme is accelerated for 15 days at 37 ℃, the PCR amplification is carried out, and the amplification stability of the amplification system is obviously superior to that of similar products in the market.

In an alternative embodiment, the DNA polymerase is a DNA polymerase belonging to family a.

In an alternative embodiment, the DNA Polymerase is at least 1 selected from the group consisting of Tth DNA Polymerase or a mutant thereof, taq DNA Polymerase or a mutant thereof, and Hawk Z05 Polymerase (e.g., hawk Z05 Fast Polymerase) or a mutant thereof.

The DNA polymerase includes, but is not limited to, a reverse transcriptase (RT enzyme) for RT-PCR, a DNA polymerase for PCR, and the like.

Such mutants include, but are not limited to, recombinant and N-terminal deletion mutants. The monoclonal antibody provided by the invention has good effect of blocking polymerase activity especially for Tth DNA polymerase and Taq DNA polymerase.

In an alternative embodiment, the monoclonal antibody is bound to the DNA polymerase by an antigen-antibody reaction at 30-60 ℃, for example at 37-50 ℃.

In an eighth aspect, the present invention further provides a method for preparing a hot start enzyme, comprising the following steps:

monoclonal antibodies were incubated with DNA polymerase.

In a preferred embodiment of the present invention, the mixing ratio of the monoclonal antibody and the DNA polymerase is: each 5U of DNA polymerase was mixed with 0.1. Mu.g to 0.5. Mu.g of monoclonal antibody.

The mixing ratio of the monoclonal antibody to the DNA polymerase affects the blocking effect of the monoclonal antibody against the DNA polymerase.

In an alternative embodiment, the monoclonal antibody is mixed with the DNA polymerase in the following ratio: each 5U of DNA polymerase was mixed with 0.3. Mu.g to 0.5. Mu.g of monoclonal antibody. At this mixing ratio, the activity of the DNA polymerase, particularly the activity of Tth DNA polymerase, is substantially completely blocked.

For example, the mixing ratio is 0.3. Mu.g of monoclonal antibody/5U of DNA polymerase, 0.4. Mu.g of monoclonal antibody/5U of DNA polymerase, and 0.5. Mu.g of monoclonal antibody/5U of DNA polymerase.

The above mixing ratio is explained as follows: 5U/. Mu.l of hot start enzyme contains 0.3-0.5. Mu.g/. Mu.l of monoclonal antibody, i.e. 5U DNA polymerase is mixed with 0.3-0.5. Mu.g of monoclonal antibody per unit volume of hot start enzyme.

In an alternative embodiment, the incubation of the monoclonal antibody with the DNA polymerase is performed at 25-37 ℃. For example, incubation is carried out at 30-37 ℃.

In an alternative embodiment, the incubation time is 0.5-2h, e.g., 0.5h,1h,1.5h,2h. The monoclonal antibody provided by the invention achieves a higher blocking effect on DNA polymerase in a shorter incubation time.

In a ninth aspect, the invention also provides the use of a hot start enzyme in a polymerase chain reaction. Including but not limited to: PCR amplification, fluorescent quantitative PCR, digital PCR, etc.

In the tenth aspect, the invention also provides application of the hybridoma cell or the monoclonal antibody in preparing the hot start enzyme.

The monoclonal antibodies described above are used to bind to DNA polymerase.

In an alternative embodiment, at least 1 monoclonal antibody against DNA polymerase binds to DNA polymerase. For example, the reduction of non-specific product production by binding of various monoclonal antibodies against DNA polymerase to DNA polymerase is also within the scope of the present invention.

In an eleventh aspect, the present invention also provides a reagent or a kit comprising the monoclonal antibody described above or the hot start enzyme described above.

In an alternative embodiment, the kit is a PCR amplification kit.

In an alternative embodiment, the above reagents further comprise stabilizers, buffers, and the like. Examples include polyvinyl alcohol, mannitol, and the like.

The reagent or the kit comprises the following application scenes: used for detecting pathogens infecting plants, pathogens infecting animals (such as customs quarantine), and target genes.

Plant pathogen detection is for example from plant bacterial diseases, plant fungal diseases, plant soil-borne diseases and/or root knot nematode diseases, animal pathogen detection is for example for detection of human and animal co-diseases, animal diseases or animal pests.

The animal epidemic is derived from at least one pathogen selected from the group consisting of: brucella animalis, mycobacterium bovis, staphylococcus aureus, rabies virus, streptococcus suis type 2, bacillus anthracis, salmonella, african swine fever virus, neocoronavirus, influenza virus, HPV virus, hepatitis b virus, human immunodeficiency virus, SARS, MERS, dengue fever virus, avian influenza, ebola and escherichia coli.

The invention has the following beneficial effects:

(1) The hybridoma cell and the monoclonal antibody secreted by the hybridoma cell have the effect of reducing non-specific amplification.

(2) The monoclonal antibody secreted by the hybridoma cell can completely block the activity of the DNA polymerase, and the activity of the DNA polymerase can be recovered by heating the blocked hot-start enzyme at high temperature for 30s-10 min.

(3) Compared with the existing hot-start enzyme, the DNA polymerase modified by the monoclonal antibody has better stability (especially thermal stability), and the normal-temperature storage time of the hot-start enzyme is obviously prolonged.

(4) The monoclonal antibody secreted by the hybridoma cells has no residual nuclease activity, i.e., no residual DNA lyase activity, no residual exonuclease activity, and no residual endonuclease activity.

(5) The monoclonal antibody secreted by the hybridoma cell can keep good low-temperature blocking effect and high-temperature release polymerization activity effect on various DNA polymerases, and has good universality.

(6) The obtained monoclonal antibody has good blocking activity, can meet the experimental requirement with low dosage, and has the advantages of low dosage and good blocking activity compared with the blocking dosage of 1ug monoclonal antibody/5U DNA polymerase in the prior art.

(7) The invention also provides a preparation method of the hybridoma cell and the monoclonal antibody, which optimizes the screening strategy, and compared with a single ELISA method for detecting serum titer, the method increases the screening of the activity of the serum blocking enzyme, and is beneficial to greatly improving the accuracy of the target animal obtained by screening, thereby increasing the possibility of screening the monoclonal antibody capable of completely blocking the activity of DNA polymerase.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the results of a block-start assay of the activity of Tth DNA polymerase with ascites fluid;

FIG. 2 is an SDS-PAGE pattern of the purified antibody;

FIG. 3 is an electrophoresis diagram showing the detection of the residual activity of DNase in an antibody;

FIG. 4 is a graph showing the results of detection of Tth DNA polymerase blocking activity by the antibody;

FIG. 5 is a graph showing the results of measurement of the thermostability of a Tth DNA polymerase blocking performance of an antibody;

FIG. 6 is a graph showing the results of the start detection of the hot-start Tth DNA polymerase at different start times;

FIG. 7 is a diagram showing the results of functional application of antibody-modified Tth DNA polymerase in a qPCR system;

FIG. 8 is a graph showing the results of detection of blocking activity of antibody against Taq DNA polymerase;

FIG. 9 is a graph showing the results of detection of the thermostability of the blocking performance of antibody against Taq DNA polymerase;

FIG. 10 is a diagram showing the results of the start detection of hot-start Taq DNA polymerase at different start times;

FIG. 11 is a graph showing the results of accelerated test detection of antibody-modified Taq DNA polymerase.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. M.j. Goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), academic Press, inc. (Academic Press, inc.), "Handbook of Experimental Immunology" ("D.M.Weir and C.C.Black well"), gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos.), "Current Protocols in Molecular Biology" (F.M.Ausubel et al., 1987), "PCR, polymerase Chain Reaction (PCR: the Polymerase Chain Reaction) (Mullis et al., 1994), and" Current Protocols in Immunology "(blood), each of which is incorporated herein by reference, cold, 1991.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Tth DNA polymerase recombinant protein purified and prepared in the laboratory is used for immunizing SPF-level female BalB/C mice of 6-8 weeks old, the immunizing dose is 50-200 mug/mouse, the immunizing amount is 10, and after three times of immunization, the titer of serum polyclonal antibody and the blocking-starting effect on enzyme activity are detected. Then, cells of mice having a serum titer of 1. And (3) obtaining a plurality of stable positive hybridoma cell strains through 3 rounds of subcloning, establishing a hybridoma cell bank, and freezing and preserving the strains. Then, the positive hybrid cell strain is identified by the following method: recovering hybridoma cells, expanding culture, preparing ascites small test, injecting 3 mice per cell strain to prepare ascites, detecting ascites titer, blocking-activating activity to enzyme, purifying ascites capable of completely blocking enzyme activity to obtain antibody small sample, and detecting the antibody small sample. The detection items comprise: antibody concentration, purity, nuclease residue detection, blocking-initiating activity detection, functionality detection, and stability detection. The screened antibody needs to completely block the enzyme activity at low temperature, separate from the binding site at high temperature, release the enzyme activity and reduce the amplification of non-specific reaction. The target antibody obtained by the invention is named by the number of the small test ascites: ttA135.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a method for preparing hybridoma cells, comprising the steps of:

(1) And (5) carrying out animal immunization and serum detection screening.

An SPF (specific pathogen free) female BalB/C mouse of 6-8 weeks old is adopted, immunogen is Tth DNA polymerase recombinant protein purified and prepared in the laboratory, the immunization dose is 50-200 mug/mouse (immunization volume is 200 mug/mouse), the number of immunized animals is 10, the animals are divided into 5 cages, and immunization is carried out by adopting a back subcutaneous multi-point method (5-7 points).

First immunization: the antigen was diluted with 1 × pbs and mixed with equal volume of freund's complete adjuvant to make the immunogen. Under the ice bath state, the immunogen is emulsified into a water-in-oil state by a high-speed homogenizer, and then animal immunization can be carried out.

And (3) secondary immunization: and performing secondary immunization after three weeks, wherein the immunogen is prepared by mixing the antigen and Freund's incomplete adjuvant in equal volume, and the other operations are the same as the primary immunization.

Three times of immunization: three immunizations were performed two weeks later, and the procedure was the same as the second immunization. One week later, tail tip vein blood is collected, the blood is placed at 4 ℃ overnight, animal serum is obtained after centrifugation, the serum titer of the animal serum and the blocking-starting activity of Tth DNA polymerase activity are respectively detected, and immune BalB/C mice which can be used for cell fusion are screened.

(2) Cell fusion was performed.

Impact immunization: the 6 BalB/C mice screened in step (1) for good blocking-priming activity were fused in triplicate, and the immunogen was diluted with 1 × pbs three days prior to fusion, i.e. i.p. injections were performed at the same dose as in step (1).

Myeloma cell preparation: recovering myeloma cells SP2/0 one week earlier, placing at 37 deg.C and 5% CO ₂ Culturing in an incubator, adjusting the cell state, and carrying out passage 36-48h before fusion, so as to ensure that the cells are in a logarithmic growth phase during fusion.

Preparing feeder cells: feeder cells in the abdominal cavity of a healthy Kunming mouse are selected to obtain growth factors secreted by the feeder cells, and one Kunming mouse can be paved into 5 96-well plates.

Cell fusion: obtaining a mouse splenocyte suspension after the impact immunization, uniformly mixing splenocytes and myeloma cells in an RPMI 1640 culture medium without serum according to the proportion of 5:1-10, centrifuging at 1200rpm for 5min, and discarding the supernatant; centrifugation at 1200rpm was carried out for 1min, and the residual RPMI 1640 was pipetted. Gently patting the scattered cell sediment with fingers, taking out 1ml of 50% PEG incubated in an incubator and a beaker containing 3/4 volume of sterile water preheated in a water bath kettle, and immersing the bottom of a centrifuge tube into the water in the beaker; sucking 1ml of 50% PEG by using a blue gun head which is cut through a tip, dropwise adding the PEG into mixed myeloma cells and splenocytes, slightly shaking a centrifugal tube while dropwise adding, and slightly blowing, sucking and uniformly mixing after adding; stop solution was added to stop. After the termination reaction, the mixture was centrifuged at 1200rpm/min for 5min, and the supernatant was discarded. Resuspending the cell sediment by using HAT culture medium, uniformly mixing, adding the cell sediment into a culture dish, and paving 10 plates in a 96-well plate at 100 mu l/hole; standing at 37 deg.C for 5% CO ₂ Culturing in an incubator.

(3) And (4) screening and cloning hybridoma cells.

Since hybridoma cells in the early stage of fusion are unstable and tend to lose the antibody-secreting ability, early cloning is required. Observing the growth state of the cells in the microporous plate under a microscope, supplementing liquid by using HAT culture medium 3 days after fusion, screening 7 days after fusion, and completely replacing liquid by using HT culture medium the day before detection and screening. And (3) coating the ELISA plate with the optimal immunogen coating concentration (the optimal coating concentration is found out by a chessboard method and is 0.25 mug/ml, and the coating volume is 50 mul), and detecting the cell culture supernatant by adopting an indirect ELISA method. Screening out OD ₄₅₀ Positive wells with higher nm values.

Hybridoma cell cloning: feeder cells were prepared the day before cloning by limiting dilution.

First cloning: HT medium, 5 cells/well, diluted, counted, plated; and (3) second cloning: 20% FBS 1640 medium, 3 cells/well, diluted, counted, plated; and (3) cloning for the third time: 20% FBS 1640 medium, 1 cell/well. For cloning, cells in 96 wells were blown up, diluted, counted, and plated.

Recording the number of cell clones in each hole 4 days after each cloning, heavily marking single clones for screening, screening 7 days after cloning, changing the total amount of the liquid in the previous day, coating an enzyme-linked immunosorbent assay (ELISA) plate by using the optimal coating concentration of immunogen, and detecting cell culture supernatant by adopting an indirect ELISA method.

Screening out OD ₄₅₀ Positive single clone wells with higher nm values. Cloning the positive monoclonals again, detecting that all the cloned holes are positive and the positive monoclonals are stable, expanding culture, freezing and preserving the seeds. Finally, a total of 180 positive hybridoma cells were obtained.

(4) Ascites lab scale preparation-functional screening of hybridoma cells was performed.

Carrying out ascites small-scale test preparation on 180 positive hybridoma cells screened in the step (3) in batches, sensitizing mice 7-10 days before cell injection, and injecting 0.5ml of liquid paraffin into abdominal cavities; resuscitating the cells on the same day, supplementing feeder cells (synchronous step (2)) to adjust the cell state, and performing amplification culture by using a 10cm cell culture dish; the cells were collected, centrifuged and resuspended in serum-free 1640 RPMI minimal medium at a cell density of 1 x 10 ⁷ Each hybridoma cell strain was injected into 3 mice, and the volume of the injected cell suspension was: 0.2ml, the injected cell volume is: 2*10 ⁶ One/only. The ascites is increased after 5-7 days, the collection is started when the mouse abdomen is tight, the collected ascites is centrifuged at 3000rpm for 10min, the upper layer of grease and the lower layer of sediment are discarded, and the middle layer is taken and stored at-20 ℃ for standby. Ascites collection frequency: daily, collection period: 7-10 days until the mice die naturally. After the collection is finished, the ascites prepared by the cells of the same strain are integrated together for identification.

Ascites identification: seal-start detection. And modifying Tth DNA polymerase by using ascites, starting the sample at 95 ℃ for 5min, simultaneously testing the sample and the sample which is not started, and comparing to judge whether the ascites has a sealing effect. If the activity of the sample after the start is obviously higher than that of the sample without the start, the ascites has a sealing effect. The sample loading of each group was 10U. Detection of ascites Tt135 can completely block Tth DNA polymerase activity, as shown in FIG. 1.

Ascites identification: and (4) subtype identification. The subtype of the antibody contained in ascites Tt135 was identified by a commercial mouse monoclonal antibody subtype detection kit, and the detection result was IgG1.

The hybridoma cells are sent and preserved in the Guangdong province microorganism strain preservation center, and the preservation addresses are as follows: the strain preservation center of the institute of microorganism research on 5 th floor of experimental building of 100 # university in the overseas district of Guangzhou City, guangdong province has the preservation numbers: GDMCC:62992 with a storage date of: 22/11/2022, with the deposited cell name: hybridoma cell TtA.

Example 2

This example carried out the purification of antibodies.

Ascites fluid prepared in example 1 was taken to select Protein a affinity chromatography packing for purification: ascites fluid was diluted with coupling buffer (20 mM PBS sodium phosphate buffer, pH 7.0) at a dilution ratio of 1: after the filtration of a 5,0.45μm filter membrane; loading the sample to an affinity purification column with good balance in advance; washing solution (20 mM PBS sodium phosphate buffer, pH 7.0) to remove unbound hetero-proteins; eluting the target protein with eluent (50 mM Gly + 70mM NaCl buffer solution, pH 3.0), adding neutralizing solution (1M Tris-HCl buffer solution, pH 9.0) to adjust pH to neutrality, dialyzing with 1 × PBS, and collecting, wherein the obtained antibody is named as TtA. The TCA-Lowry method is used for detecting the concentration of the antibody, the SDS-PAGE is used for detecting the purity of the antibody, and the electrophoresis result is shown in figure 2.

FIG. 2 shows that the purified antibody has high purity and can be used in subsequent detection experiments.

Experimental example 1

And detecting the residual activity of the DNase in the antibody.

The antibody and different DNAs are incubated together, the system is prepared as shown in Table 1, electrophoresis detection is carried out, the change of an electrophoresis band is observed, and whether the antibody has the residual activity of the DNase (comprising the activities of DNA lytic enzyme, exonuclease and endonuclease) is judged.

The results are shown in fig. 3, wherein lane 1 in fig. 3 is marker,

lanes

2, 3 and 4 are dnase detection lanes,

lanes

5, 6 and 7 are endonuclease detection lanes, and

lanes

8, 9 and 10 are exonuclease detection lanes, wherein

lanes

2, 3, 5, 6, 8 and 9 are lanes in which antibodies and DNA are mixed and incubated, and

lanes

4, 7 and 10 are negative controls. The results show that the antibody prepared by the present invention has no DNA lyase, no exonuclease activity residue, and no endonuclease activity residue.

TABLE 1 reaction System preparation Table

Enzyme residue detection item	Test system and standard
		DNA lytic enzyme	20. Mu.l of the reaction system was reacted with 5. Mu.g of antibody and 0.2. Mu.g of lambda DNA at 37 ℃ for 16 hours, and the DNA band was not changed by agarose gel electrophoresis.
Endonuclease	20. Mu.l of 5. Mu.g of antibody and 0.2. Mu.g of Supercoiled pUC18 DNA in the reaction system were reacted at 37 ℃ for 16 hours, and the DNA band was electrophoresed on agarose gel No change occurred.
		Exonuclease	20. Mu.l of 5. Mu.g of antibody and 0.2. Mu.g of Hind-III digest. Lamda.DNA in the reaction system were incubated at 37 ℃ for 16 hours and the DNA bands were electrophoresed on agarose gel No change occurred.

Experimental example 2

The antibodies of this experimental example were examined for the blocking-initiating effect of Tth DNA polymerase activity.

The TtA antibody and the Tth DNA polymerase are mixed in different proportions and incubated for 1h at 37 ℃ to allow the TtA antibody to form a complex with the Tth DNA polymerase, i.e., the Tth DNA polymerase is hot started.

The antibody-antigen mixing ratio was as follows:

the antibody concentration in 5U/. Mu.l of the hot-started Tth DNA polymerase was 0.1. Mu.g/. Mu.l, and the modification degree was 0.1.

The antibody concentration in 5U/. Mu.l of the hot-started Tth DNA polymerase was 0.2. Mu.g/. Mu.l, and the modification degree was 0.2.

The antibody concentration in 5U/. Mu.l of the hot-started Tth DNA polymerase was 0.3. Mu.g/. Mu.l, and the modification degree was 0.3.

The antibody concentration in 5U/. Mu.l of the hot-started Tth DNA polymerase was 0.4. Mu.g/. Mu.l, and the modification degree was 0.4.

The antibody concentration in 5U/. Mu.l of the hot-started Tth DNA polymerase was 0.5. Mu.g/. Mu.l, and the modification degree was 0.5.

(1) Detection of sealing performance:

performing qPCR reaction by using molecular beacon as template and primer and the prepared hot-start Tth DNA polymerase as amplification polymerase, wherein the negative control group uses unmodified Tth DNA polymerase as amplification polymerase, and the blank control group uses H ₂ O instead of hot start Tth DNA polymerase. The qPCR reaction system preparation table is shown in table 2.

TABLE 2 qPCR reaction System preparation Table

Reagent	Amount of addition
		Molecular beacons	2.5μl
dNTP(10mM)	0.5μl
		10 X Buffer for qPCR	2.0μl
Hot-start (unmodified) Tth DNA polymerase	2.0μl
		H ₂ O	Make up to 20 μ l

The reaction procedure is as follows: at 40 ℃ for 5s,200 cycles.

The experimental results are shown in FIG. 4, and it can be seen that the negative control group can be normally amplified, which indicates that the reaction system and operation are normal; the hot-start Tth DNA polymerases with different modification degrees have different blocking effects, and when the modification degree is 0.1-0.2, the activity of the Tth DNA polymerase is greatly reduced, which indicates that the activity of the Tth DNA polymerase can be effectively blocked when the modification degree is 0.1-0.2; when the modification degree is more than 0.3, the amplification line is the same as that of the negative control group, which indicates that the activity of Tth DNA polymerase is basically completely blocked when the modification degree is more than 0.3; fluorescence value of blank control group is background value.

(2) And (3) detecting the stability of the sealing performance:

selecting hot-start Tth DNA polymerase with modification degree of 0.3, heating the hot-start Tth DNA polymerase at 50 deg.C/55 deg.C/60 deg.C for 1 hr, respectively, detecting sealing performance (reaction system shown in Table 2), using unmodified Tth DNA polymerase as amplification polymerase in negative control group, and H in blank control group ₂ O instead of hot start Tth DNA polymerase.

The experimental results are shown in FIG. 5, and it can be seen from FIG. 5 that the negative control group can be normally amplified, indicating that both the reaction system and the operation are normal; the hot-start Tth DNA polymerase heated at different temperatures is basically the same as the unheated amplification line, which shows that the blocking effect of the hot-start enzyme is very stable below 60 ℃; fluorescence value of blank control group is background value.

(3) Start-Up detection of Hot Start Tth DNA polymerase

Selecting hot start Tth DNA polymerase with modification degree of 0.3, heating the hot start Tth DNA polymerase at 95 deg.C for 30s/1min/5min/10min, respectively, detecting sealing performance after heating (reaction system is shown in Table 2), using unmodified Tth DNA polymerase as amplification polymerase in negative control group, and H in blank control group ₂ O instead of hot start Tth DNA polymerase.

The experimental results are shown in FIG. 6, and it can be seen that the negative control group can be normally amplified, which indicates that the reaction system and operation are normal; the activity of the closed Tth DNA polymerase is recovered by respectively heating for 30s/1min/5min/10min at the temperature of 95 ℃, and the activity basically equivalent to that of a negative control can be recovered, which indicates that the activity of the hot-start Tth DNA polymerase can be recovered by heating for more than 30s at the temperature of 95 ℃; fluorescence value of blank control group is background value.

Experimental example 3

And (3) detecting the functional application of the antibody to Tth DNA polymerase in a qPCR system.

The monoclonal antibody TtA prepared by the invention is prepared into a qPCR amplification system, and the influence of the antibody on the PCR effect is detected. Genome templates were grouped as in table 3, reaction system was formulated as in table 4, amplification procedure: at 50 ℃ for 2min;95 ℃ for 5min;50cycles (95 ℃,10s, 55 ℃,40 s). The different lines show the graphs of qPCR amplifications performed using different amounts of genome as templates using hot-start Tth DNA polymerase with a modification degree of 0.3 and unmodified Tth DNA polymerase, and three template gradients were performed using both hot-start Tth DNA polymerase and unmodified Tth DNA polymerase.

The results are shown in FIG. 7: in an amplification system, the amplification effect of the Tth DNA polymerase modified by the antibody TtA prepared by the invention is obviously better than that of unmodified enzyme.

TABLE 3 grouping of genomic templates

Form panel	Human genome template
		Template arrangement	0.125 ng/μl,2T ； 0.0125 ng/μl,2T ； 0.00125 ng/μl,4T 。 10μl/T
Procedure	50℃ ， 2min ； 95℃ ， 5min ； 50cycles （ 95℃ ， 10s ； 55℃ ， 40s ）

TABLE 4 reaction system preparation Table

Reagent	Amount of addition
		DNA quadruple primer probe	0.5μl
dNTP （ 16.67mM)	0.3μl
		UNG(1U/μl ）	0.25μl
12.5×Buffer for qPCR	2μl
		Hot Start (unmodified) Tth DNA polymerase (5U/. Mu.l)	0.25μl
H ₂ O	Make up to 15 μ l

Experimental example 4

This example was carried out to examine the blocking-promoting effect of the antibody on the Taq DNA polymerase activity.

The TtA antibody and Taq DNA polymerase are mixed in different proportions and incubated at 37 ℃ for 1h to allow the TtA antibody and Taq DNA polymerase to form a complex, i.e., the Taq DNA polymerase is hot-started.

The antibody-antigen mixing ratio was as follows:

the antibody concentration in hot-started 5U/. Mu.l Taq DNA polymerase was 0.1. Mu.g/. Mu.l, and the modification degree was designated as 0.1.

The antibody concentration in hot-started 5U/. Mu.l Taq DNA polymerase was 0.2. Mu.g/. Mu.l, and the modification degree was recorded as 0.2.

The antibody concentration in hot-started 5U/. Mu.l Taq DNA polymerase was 0.3. Mu.g/. Mu.l, and this was designated as a hot-started Taq DNA polymerase with a modification degree of 0.3.

The antibody concentration in hot-started 5U/. Mu.l Taq DNA polymerase was 0.4. Mu.g/. Mu.l, and the modification degree was recorded as 0.4.

The antibody concentration in hot-started 5U/. Mu.l Taq DNA polymerase was 0.5. Mu.g/. Mu.l, and the modification degree was recorded as 0.5 of hot-started Taq DNA polymerase.

(1) And (3) detection of sealing performance:

taking molecular beacon as a template and a primer, taking the prepared hot start Taq DNA polymerase as amplification polymerase to carry out qPCR reaction, taking unmodified Taq DNA polymerase as amplification polymerase in a negative control group, and taking H as a blank control group ₂ O instead of Hot Start Taq DNA polymerase. The qPCR reaction system preparation table is shown in table 5.

TABLE 5 formulation of qPCR reaction system

Reagent	Amount of addition
		Molecular beacons	2.5μl
dNTP(10mM)	0.5μl
		10 X Buffer for qPCR	2.0μl
Hot start (unmodified) Taq DNA polymerase	2.0μl
		H ₂ O	Make up to 20 μ l

The reaction procedure is as follows: 40 ℃ for 5s,200 cycles.

The experimental results are shown in FIG. 8, and it can be seen that the negative control group can be normally amplified, indicating that the reaction system and operation are normal; the hot start Taq DNA polymerases with different modification degrees have different blocking effects, when the modification degree is 0.1-0.2, the activity of the Taq DNA polymerase is greatly reduced, which indicates that the activity of the Taq DNA polymerase can be effectively blocked when the modification degree is 0.1-0.2; when the modification degree is more than 0.3, the amplification line is the same as that of the negative control group, which indicates that the activity of Taq DNA polymerase is basically completely closed when the modification degree is more than 0.3; fluorescence value of blank control group is background value.

(2) And (3) detecting the stability of the sealing performance:

selecting hot start Taq DNA polymerase with modification degree of 0.3, heating the hot start Taq DNA polymerase at 50 ℃/55 ℃/60 ℃ for 1H respectively, detecting the sealing performance after heating (the reaction system refers to table 5), taking unmodified Taq DNA polymerase as an amplification polymerase in a negative control group, and taking H as a blank control group ₂ O instead of Hot Start Taq DNA polymerase.

The experimental results are shown in FIG. 9, which shows that the negative control group can be amplified normally, indicating that the reaction system and operation are normal; the hot start Taq DNA polymerase heated at different temperatures is basically the same as the unheated amplification line, which shows that the blocking effect of the hot start enzyme is very stable below 60 ℃; fluorescence value of blank control group is background value.

(3) Hot start Taq DNA polymerase initiation assays

Selecting hot start Taq DNA polymerase with a modification degree of 0.3, heating the hot start Taq DNA polymerase for 30s/1min/5min/10min at 95 ℃, respectively, detecting the sealing performance after heating (the reaction system refers to table 5), taking unmodified Taq DNA polymerase as an amplification polymerase in a negative control group, and taking H as a blank control group ₂ O instead of Hot Start Taq DNA polymerase.

The experimental results are shown in FIG. 10, which shows that the negative control group can be amplified normally, indicating that the reaction system and operation are normal; the activity of the closed Taq DNA polymerase is recovered by respectively heating for 30s/1min/5min/10min at the temperature of 95 ℃, and the activity of the closed Taq DNA polymerase can basically recover to the activity equivalent to that of a negative control, which indicates that the activity of the hot-start Taq DNA polymerase can be recovered by heating for more than 30s at the temperature of 95 ℃; fluorescence value of blank control group is background value.

Example 5

And (3) detecting the function application of the antibody to Taq DNA polymerase in a PCR system.

The monoclonal antibody TtA prepared by the invention is prepared into a PCR amplification system, the influence of the antibody modified Taq DNA polymerase on the PCR amplification effect is detected, and the similar products in the market are compared. Test method (single tube stability test): preparing components such as PCR reaction Buffer, amplification enzyme, primer probe and the like into a single-tube reagent, uniformly mixing, standing at 37 ℃ for 15 days, and comparing the amplification performance of the accelerated single-tube reagent by taking a single tube of a corresponding reaction system stored at-20 ℃ as a reference. The amplification system is prepared as shown in Table 6. And (3) amplification procedure: at 50 ℃ for 2min;95 ℃ for 5min;50cycles (95 ℃,10s, 55 ℃,40 s).

The results are shown in FIG. 11: in an amplification system, a single tube of a Taq enzyme preparation system modified by TOYOBO Hot Start rTaq DNA Polymerase (TAP-211) of the same product in the market is accelerated by 15d at 37 ℃ to reduce Rn value amplified in a Vic channel by over 50 percent, and CT value is lagged, and the stability of the single tube of the Taq enzyme preparation system modified by the antibody TtA prepared by the invention is obviously superior to that of the same product in the market.

TABLE 6 amplification System configuration Table

System of	2×Robustart Premix-UNG （ Probe qPCR ）
		Form panel	HBV-MLN pure template
Template arrangement	1E4 IU/ml ， 2T ； 1E3 IU/ml ， 2T ； 50 IU/ml ， 4T 。

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A hybridoma cell secreting a monoclonal antibody against DNA polymerase, which is deposited at the guangdong province collection of microbial cultures at the following addresses: the strain preservation center of the institute of microorganism research on 5 th floor of experimental building of 100 # university in the overseas district of Guangzhou City, guangdong province has the preservation numbers: GDMCC:62992 with a storage date of: 22/11/2022, deposited under the cell name: hybridoma cell TtA.

2. A monoclonal antibody secreted by the hybridoma cell of claim 1, wherein the monoclonal antibody specifically binds to a DNA polymerase.

3. A hot start enzyme comprising a DNA polymerase modified with an antibody, wherein the antibody is the monoclonal antibody of claim 2, wherein the monoclonal antibody binds to the DNA polymerase by an antigen-antibody reaction; the DNA polymerase is at least one selected from Tth DNA polymerase or mutant thereof, taq DNA polymerase or mutant thereof.

4. A method for preparing a hot-start enzyme according to claim 3, comprising the steps of:

incubating the monoclonal antibody with the DNA polymerase.

5. The method for preparing hot start enzyme according to claim 4, wherein the mixing ratio of the monoclonal antibody and the DNA polymerase is: each 5U of DNA polymerase was mixed with 0.1. Mu.g to 0.5. Mu.g of monoclonal antibody.

6. The method for preparing hot-start enzyme according to claim 5, wherein the mixing ratio of the monoclonal antibody to the DNA polymerase is: each 5U of DNA polymerase was mixed with 0.3. Mu.g to 0.5. Mu.g of monoclonal antibody.

7. The method for producing a hot-start enzyme according to claim 4, wherein the incubation of the monoclonal antibody with the DNA polymerase is performed at 25 to 37 ℃; the incubation time is 0.5-2h.

8. Use of a hot start enzyme according to claim 3 or prepared according to the method of any one of claims 4 to 7 in a polymerase chain reaction.

9. Use of a hybridoma cell according to claim 1 or a monoclonal antibody according to claim 2 for the preparation of a hot start enzyme; the monoclonal antibody is used for combining with DNA polymerase, and the DNA polymerase is at least one selected from Tth DNA polymerase or a mutant thereof, taq DNA polymerase or a mutant thereof.

10. A reagent or kit comprising the monoclonal antibody of claim 2 or the hot start enzyme of claim 3.