CN117659200A - Pfu DNA polymerase antibody combination and application thereof - Google Patents

Abstract

The invention provides a Pfu DNA polymerase antibody combination, including Pfu DNA polymerase monoclonal antibody R9C8 and Pfu DNA polymerase monoclonal antibody F10G6; the heavy chain variable region sequence of the monoclonal antibody R9C8 is as SEQ ID NO: As shown in 1, the light chain variable region sequence is shown in SEQ ID NO:2; the heavy chain variable region sequence of the monoclonal antibody F10G6 is shown in SEQ ID NO:3, and the light chain variable region sequence is shown in As shown in SEQ ID NO:4. This combination has better 5'-3' end polymerase activity of blocking Pfu DNase in a wide temperature range (0 ~ 70℃) before the pre-denaturation stage.

Description

A Pfu DNA polymerase antibody combination and its application

Technical field

The invention relates to a Pfu DNA polymerase antibody combination and its application, belonging to the technical field of Pfu DNA polymerase.

Background technique

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It has the characteristics of strong specificity, high sensitivity, rapidity, simplicity and good reproducibility. It is one of the most basic and important tools in molecular biology research. Its principle is to amplify DNA molecules in vitro under the catalysis of DNA polymerase, using parent strand DNA as a template and primers as starting points through cyclic steps such as denaturation, annealing and extension, thereby achieving DNA replication.

Pfu DNA polymerase (Pfu enzyme for short) is a DNA polymerase isolated by scientists from Pyrococcus furiosus (Pfu). The enzyme contains two protein subunits (P45 and P50) and is a polymer. Its molecular weight is 90kda and it has excellent thermal stability. Unlike TaqDNA polymerase, this enzyme has both 5'-3' polymerase activity and 3'-5' exonuclease activity. The main function of the 3'-5' exonuclease activity is proofreading, allowing instant identification. And excise mismatched nucleotides, which gives the Pfu enzyme extremely high fidelity. Due to its high fidelity and high stability, Pfu enzyme has become one of the most widely used DNA polymerases.

During the PCR process, Pfu enzyme, like other DNA polymerases, still has certain polymerase activity under low temperature conditions, resulting in non-specific amplification and the formation of primer-dimers. Currently, there are many ways to achieve hot start, such as genetic mutation, physical isolation, chemical modification, and nucleic acid primer modification of enzymes. However, these methods all have unfavorable factors, such as incomplete blocking of enzyme activity, affecting the complexity of PCR, affecting the processability and fidelity of the enzyme, or the method is relatively cumbersome.

In view of the shortcomings of the above related technologies, using monoclonal antibodies to modify Pfu enzyme is a relatively better method. Pfu enzyme antibody is a hot-start PCR anti-Pfu enzyme antibody, which inhibits DNA polymerase activity after binding to Pfu enzyme. During PCR amplification, the Pfu enzyme antibody before high-temperature denaturation binds to the Pfu enzyme to inhibit DNA polymerase activity, which can effectively inhibit non-specific annealing of primers and non-specific amplification caused by primer dimers under low-temperature conditions, and improve the efficiency of amplification. Specificity and target DNA yield. However, in existing PCR, only one Pfu DNA polymerase antibody is usually used. If the temperature is above 70°C, the blocking efficiency of the Pfu DNA polymerase antibody on Pfu DNA polymerase decreases significantly, resulting in some non-specific amplification.

Contents of the invention

The invention provides a Pfu DNA polymerase antibody combination and its application, which can effectively solve the above problems.

The present invention is implemented as follows:

A Pfu DNA polymerase antibody combination, including Pfu DNA polymerase monoclonal antibody R9C8 and Pfu DNA polymerase monoclonal antibody F10G6; the heavy chain variable region sequence of the monoclonal antibody R9C8 is shown in SEQ ID NO: 7 , the light chain variable region sequence is shown in SEQ ID NO: 8; the heavy chain variable region sequence of the monoclonal antibody F10G6 is shown in SEQ ID NO: 17, and the light chain variable region sequence is shown in SEQ ID NO. :18.

In some embodiments, the CDR1 sequence of the heavy chain variable region sequence of the monoclonal antibody R9C8 is as shown in SEQ ID NO: 1, the CDR2 sequence is as shown in SEQ ID NO: 2, and the CDR3 sequence is as shown in SEQ ID NO: 3 Show,

In some embodiments, the CDR1 sequence of the light chain variable region sequence of the monoclonal antibody R9C8 is as shown in SEQ ID NO: 4, the CDR2 sequence is as shown in SEQ ID NO: 5, and the CDR3 sequence is as shown in SEQ ID NO: 6 Show.

In some embodiments, the CDR1 sequence of the heavy chain variable region sequence of the monoclonal antibody F10G6 is as shown in SEQ ID NO: 11, the CDR2 sequence is as shown in SEQ ID NO: 12, and the CDR3 sequence is as shown in SEQ ID NO: 13 Show.

In some embodiments, the CDR1 sequence of the light chain variable region sequence of the monoclonal antibody F10G6 is as shown in SEQ ID NO: 14, the CDR2 sequence is as shown in SEQ ID NO: 15, and the CDR3 sequence is as shown in SEQ ID NO: 16 Show.

In some embodiments, the heavy chain sequence of the monoclonal antibody R9C8 is shown in SEQ ID NO:9, and the light chain sequence is shown in SEQ ID NO:10.

In some embodiments, the heavy chain sequence of the monoclonal antibody F10G6 is shown in SEQ ID NO: 19, and the light chain sequence is shown in SEQ ID NO: 20.

In some embodiments, the mass ratio of the Pfu DNA polymerase monoclonal antibody R9C8 and the Pfu DNA polymerase monoclonal antibody F10G6 is 0.5-1.5:1.

Application of the above-mentioned Pfu DNA polymerase antibody combination in reducing non-specific amplification of Pfu DNA polymerase.

In some embodiments, the mass ratio of the Pfu DNA polymerase antibody combination to Pfu DNA polymerase is 1.5-2.5:1.

The beneficial effects of the present invention are:

The invention provides a Pfu DNase monoclonal antibody combination, including R9C8 antibody and F10G6 antibody, which play a synergistic effect. This combination has a wide temperature range before the pre-denaturation stage. Blocks the 5'-3' polymerase activity of Pfu DNA polymerase within (0-70℃), and its blocking efficiency of blocking the 5'-3' polymerase activity of Pfu DNA polymerase at 70℃ is more than 97%. This antibody The combination in PCR greatly reduces non-specific amplification.

Description of 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 introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a graph of polymerase activity blocking efficiency of Pfu enzyme antibody combination.

Figure 2 is a graph of the polymerase activity blocking efficiency of Pfu enzyme R9C8 antibody.

Figure 3 is a graph of the polymerase activity blocking efficiency of Pfu enzyme F10G6 antibody.

Figure 4 is a nucleic acid electrophoresis chart of PCR results of antibody group enzymes, single antibody enzymes (R9C8 antibody enzyme and F10G6 antibody enzyme) and PfuDNA polymerase.

Detailed ways

In order to make the purpose, 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 in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Accordingly, the following detailed description of embodiments of the invention provided in the appended drawings is not intended to limit the scope of the claimed invention, but rather to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include one or more of these features. In the description of the present invention, "plurality" means two or more than two, unless otherwise explicitly and specifically limited.

Example 1

Pfu DNA polymerase preparation

1. Acquisition, synthesis and vector construction of Pfu DNA polymerase gene

According to the sequence of Pfu DNA polymerase found by NCBI (NCBI: NC_003413.1), the present invention introduces an Nde I enzyme cleavage site at the N terminus of the gene, and introduces a stop codon and an Xho I enzyme cleavage site at the C terminus to synthesize Gene (Shanghai Sangon Biosynthesis). The gene and pET28a vector were double-digested with Nde I and Xho I restriction endonucleases respectively, and then T4 DNA ligase was used to construct the pET28a-Pfu vector.

2. Construction and expression of Pfu DNA polymerase recombinant bacteria

The constructed pET28a-Pfu vector was transformed into E. coli BL21 (DE3) strain (purchased from Shanghai Sangon Biotech), spread on LB plates, cultured overnight, and single colonies were picked. Inoculate into 10mL LB liquid culture medium, add Kan antibiotics to a final concentration of 1mM, culture overnight at 37°C, 250rpm, transfer to 200mL LB culture medium at a ratio of 1:100 the next day, add Kan antibiotics to a final concentration of 1mM, 37 ℃, 250rpm culture until the OD value is 0.6-0.8, add isopropylthiogalactopyranoside with a final concentration of 0.01mM/L-0.2mM/L, and induce expression at 25℃ for 6-8h. Centrifuge the bacterial liquid to collect the bacterial cells. Centrifuge conditions: 4°C, 8000 rpm for 10 minutes.

3. Purification of Pfu DNA polymerase

(1) Mix the bacterial cells and BufferA (Tirs-HCl 20mM, sodium chloride 500mM, imidazole, 10mM, adjust pH to 7.4) at a mass ratio of 1:10, and crush by ultrasonic in an ice bath. Crushing conditions: power 300W, ultrasonic 3S, Pause for 8S and break for 15min;

(2) Centrifuge at 12,000 rpm for 30 minutes at 4°C, collect the supernatant, filter the supernatant with a 0.22 μm filter membrane, and load it onto the Ni chromatography column;

(3) Use BufferB (Tirs-HCl 20mM, sodium chloride 500mM, imidazole, 20mM, adjust pH to 7.4) to wash the impurity protein;

(4) Use BufferC (Tirs-HCl 20mM, sodium chloride 500mM, imidazole, 200mM, DTT 8mM, adjust pH to 7.4) to elute Pfu DNA polymerase;

(5) Ultrafiltrate the purified Pfu DNA polymerase, change the liquid, concentrate, and store it in a storage Buffer (Tris-HCl (pH7.4) 20mM, KCl 100mM, DTT 1mM, 0.5% Tween 20, 0.1mM EDTA, glycerol 50% (v/v).

Example 2

Monoclonal antibody preparation

The present invention prepares two monoclonal antibodies of Pfu DNA polymerase, namely R9C8 and F10G6.

The antibody preparation process is as follows:

The reagents or kits involved in this application and their sources are as follows:

Freund's Adjuvant, Complete (Cat. No. 77140, Thermo Fisher); Freund's Adjuvant, Incomplete (Cat. No. 77145, Thermo Fisher); HATMediaSupplement (50×) (Cat. No.: 21060017, Thermo Fisher); HT MediaSup plement (50×) (Cat. No. H0111067030, Thermo Fisher); PEG (Cat. No. P7181, Sigma Company); RPMI 1640 (Cat. No. L210KJ, Shanghai Yuanpei Biotechnology); Fetal Bovine Serum FBS (C04001-500 (Product No.: L310KJ, Shanghai Yuanpei Biotech); DMEM (Product No.: L310KJ, Shanghai Yuanpei Biotech); Penicillin-St reptomycin (Product No.: 15140122, gibco); HRP-labeled goat anti-mouse antibody (Product No.: D110087, Shanghai Sangon Biotechnology); Protein A Resin (Cat. No. SA023010, Changzhou Tiandi Renhe Biotechnology Co., Ltd.).

1. Mouse immunization:

Use Pfu DNA polymerase to immunize 8-10 week old, healthy Balb/c female mice weighing about 20 g multiple times. The first immunization is a subcutaneous injection of 50 μg Pfu DNA polymerase and Freund's complete adjuvant on the back of the mouse. The immunogen obtained after mixing the volume; a second immunization was performed two weeks later, and each mouse was injected subcutaneously on the back with 50 μg of Pfu DNA polymerase and an equal volume of Freund's incomplete adjuvant. The immunogen was obtained; a third immunization was performed two weeks later. , the immunogen obtained by mixing 50 μg Pfu DNA polymerase and Freund's incomplete adjuvant in equal volumes was injected subcutaneously on the back of each mouse; two weeks later, the immunity was strengthened, and each mouse was injected intraperitoneally with 50 μg Pfu DNA polymerase and equal volumes of normal saline. The immunogen obtained after mixing.

2. Cell fusion: Start on the third day after the last immunization of mice. Take BALB/c mice on the third day after the last immunization, remove the eyeballs to collect blood, and isolate the mouse serum as a positive control for antibody detection. At the same time, the mice are killed by cervical dislocation, their spleens are taken, and a spleen cell suspension is prepared. Resuscitate myeloma cells two weeks in advance (make sure the cells are in logarithmic growth phase when used) to obtain myeloma cells. Mouse peritoneal macrophages were obtained the day before fusion and added to a 96-well plate for culture to obtain a cell plate containing feeder cells. Using the PEG-mediated fusion method, take the spleen cell suspension and myeloma cell suspension at a cell number ratio of 5:1, mix them in serum-free DMEM culture medium, centrifuge at 1200 rpm for 5 minutes, remove the supernatant, and use your fingers to Flick the bottom of the centrifuge tube to loosen and mix the two cells. Place it in a beaker filled with 37°C water and keep it warm. Add 1mL of 50% PEG (pH8.0) fused cells within 1 minute, shake while adding, and finish adding. Then let it stand for 30 seconds, add serum-free DMEM medium to terminate fusion, centrifuge at 1000 rpm for 5 minutes, resuspend the pellet in HAT medium, and aliquot it into a 96-well cell plate containing feeder cells, and culture the cells at 37°C and 5% CO2. Cultured in the box. Among them, preparing 500mL HAT medium requires 100mL FBS, 5mL penicillin-streptomycin (Penicillin-Streptomycin), 10mL HAT Media Supplement, and 385mL DMEM.

3. Screening of hybridoma cells: Cultivate in the cell culture incubator until the 7th day. When the fused cells cover 10%-50% of the bottom of the well, screen the positive wells using conventional indirect ELISA method. Use Pfu DNA polymerase to coat the plate and detect the hybridoma cell culture supernatant. The secondary antibody is an enzyme-labeled goat anti-mouse antibody. The mouse serum isolated in step 2 is used as a positive control to screen out positive hybridoma cells with higher antibody titers.

4. Hybridoma cell cloning: The day before cloning, prepare feeder cells according to step 2 and plate them; use a pipette to mix the positive hybridoma cells in the wells to be cloned, and dilute the cells in the wells with HT medium to 1 cell in each well; culture at 37°C and 5% _CO2 for 7-10 days in a humidified environment. Antibodies can be detected when clones are visible to the naked eye. Observe under an inverted microscope and mark the wells where only a single clone grows. Finally, preliminary Screen and obtain monoclonal antibody hybridoma cell lines.

5. Preparation of monoclonal antibody ascites: Inject 0.5 ml of liquid paraffin intraperitoneally into Balb/c mice, and 10 days later inoculate 1×10 ⁶ of the selected hybridoma cell lines into the abdominal cavity of Balb/c mice. After about 10 days, the mice's abdomen will begin to swell. The mice will be sacrificed by cervical vertebra dislocation, soaked and disinfected in 75% alcohol for 5 minutes, and one-time ascites extraction will be performed.

6. Monoclonal antibody purification: centrifuge at 3000 rpm for 10 minutes. The colorless and transparent middle layer is ascites. Mix saturated (NH ₄ ) ₂ SO ₄ with ascites at a ratio of 1:1, mix thoroughly, centrifuge at 10,000 rpm for 10 min, discard the supernatant, redissolve the precipitate in a PB solution with pH 7.0 and a concentration of 20 mM, and pass through a protein A column. , eluted with Gly-HCl at pH 2.7 and concentration of 100mM, and the eluent was immediately neutralized with Tris-HCl at pH 9.0 and concentration of 1M. Subsequently, dialyze for 6 hours at 4°C and replace the storage solution with PBS solution to obtain purified monoclonal antibodies R9C8 and F10G6 of Pfu DNA polymerase.

After sequencing, the specific information is as follows:

The amino acid sequence of R9C8 is as follows:

Heavy chain CDR1 sequence: YCRT (SEQ ID NO: 1)

Heavy chain CDR2 sequence: QGHDNETDKPRILS (SEQ ID NO: 2)

Heavy chain CDR3 sequence: GLSNKIHDL (SEQ ID NO: 3)

Light chain CDR1 sequence: QSLYANDS (SEQ ID NO: 4)

Light chain CDR2 sequence: YSTLY (SEQ ID NO: 5)

Light chain CDR3 sequence: QQWTLPT (SEQ ID NO: 6)

The heavy chain variable region sequence is: QVQLQQSGAELVKPGTSVKLSCKASGYNFI YCRTWVKLRPGQGLEWIGQGHDNETDKPRILSKATLTVDKSSSTAYMQL SGLASADSAVYYCTR GLSNKIHDLWGQGTTLTVSS (SEQ ID NO:7) The light chain variable region sequence is:

DVVMTQSTPSLSVSLGDRVTISCQSLYANDSWYQQKPGTVPKLLIY QSLYANDSRVPSRFSASGSGTDFSLTISNLEQEDFATYFCYSTLYFGGGTK LEIK(SEQ ID NO:8)

The heavy chain sequence is:

Question KKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ IDNO:9)

Light chain sequence:

DVVMTQSTPSLSVSLGDRVTISCQSLYANDSWYQQKPGTVPKLLIYQSLYANDSRVPSRFSASGSGTDFSLTISNLEQEDFATYFCYSTLYFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(S EQ ID NO:10)

The amino acid sequence of F10G6 is as follows:

Heavy chain CDR1 sequence: YTMH (SEQ ID NO: 11)

Heavy chain CDR2 sequence: WAPSINTNYEKFKD (SEQ ID NO: 12)

Heavy chain CDR3 sequence: HGRKTFDY (SEQ ID NO:13)

Light chain CDR1 sequence: ASQTDSYL (SEQ ID NO:14)

Light chain CDR2 sequence: YSYLE (SEQ ID NO: 15)

Light chain CDR3 sequence: QQKENPT (SEQ ID NO:16)

The heavy chain variable region sequence is: QVQLQQSGAELVKPGASVKLSCKMSAYNFT YTMHWVKQRPGQGLEWIGWAPSINTNYEKFKDKATLSVDTSSNTAYMQ LSSLTSEDSALYYCARHGRKTFDYWGQGTTLTVSA (SEQ ID NO: 17) The light chain variable region sequence is:

DIVMTQTTASLAVSLGDRATISCASQTDSYLWYQQKPGQPPKLLIKY SYLEGVPSRFSGSGSGTDYSLTIHPVEQEDIATYYCQQKENPTFGGGTKL EIK(SEQ ID NO:18)

The heavy chain sequence is:

Question PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSY SCSVVHEGLHNHHTTKSFSRTPGK(SEQ IDNO:19)

The light chain sequence is:

DIVMTQTTASLAVSLGDRATISCASQTDSYLWYQQKPGQPPKLLIKYSYLEGVPSRFSGSGSGTDYSLTIHPVEQEDIATYYCQQKENPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC( SEQ ID NO:20)

Mix R9C8 and F10G6 at a mass ratio of 1:1 to form an antibody combination.

Example 3

Preparation of antibody enzyme and Pfu enzyme

Take the antibody combination with a concentration of 5 mg/mL, mix it with Pfu DNA polymerase with a concentration of 0.5 mg/mL at a mass ratio of 2:1, and incubate at 37°C for 30 minutes to form the antibody group enzyme.

Take the antibody R9C8 at a concentration of 5 mg/mL, mix it evenly with Pfu DNA polymerase at a concentration of 0.5 mg/mL at a mass ratio of 2:1, and incubate at 37°C for 30 minutes, which is the R9C8 antibody enzyme.

Take the antibody F10G6 at a concentration of 5 mg/mL, mix it evenly with Pfu DNA polymerase at a concentration of 0.5 mg/mL at a mass ratio of 2:1, and incubate at 37°C for 30 minutes, which is the F10G6 antibody enzyme.

Take another Pfu DNA polymerase with a concentration of 0.5mg/mL and add the same volume of antibody storage buffer, mix evenly, and incubate at 37°C for 30 minutes to become Pfu enzyme.

Example 4

5’-3’ polymerase activity blocking assay

hairpin oligonucleotide

TAGCGAAGGATGTGAACCTAATCCCTGCTCCCGCGGCCGATCTGCCG GCCGCGG (SEQ ID NO: 21), diluted to 100 μmol/L.

Prepare 10×Pfu buffer: 250mmol/L Tris-HCl, 50mmol/L (NH ₄ ) SO ₄ , 500mmol/LKCl, 1% (volume ratio) Tritonx-100, pH 8.8 (25°C), 25mmol/L MgCl ₂ , 25mmol/L dNTP.

Prepare according to the formula in Table 1 below. All operations are performed on ice. Each experiment has three replicates:

Table 1

Set the temperature program on the fluorescence quantitative PCR machine:

Amplification temperature is 37℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃; amplification time is 16s; 120 cycles; fluorescence acquisition channel; FAM.

Preheat the fluorescence quantitative PCR instrument for half an hour according to the above temperatures, place the eight-tube PCR tubes added with the reaction solution into the fluorescence quantitative PCR instrument, and start the polymerization reaction.

After the reaction is completed, the difference between the fluorescence value at 120 cycles and the initial value is calculated based on the fluorescence quantitative PCR data. The blocking effect of the 5’-3’ polymerase activity of the detection enzyme = 1-the difference in fluorescence before and after the test antibody enzyme/the difference in fluorescence value before and after the Pfu enzyme.

The results are shown in Figure 1-3. The R9C8 antibody can block the 5'-3' polymerase activity of Pfu DNA polymerase at 65°C, and the blocking efficiency is above 95%; the F10G6 antibody can block the 5'-3' polymerase activity of Pfu DNA polymerase at 60°C. 3' polymerase activity, and the blocking efficiency is more than 98%; at 70℃, the blocking efficiency of R9C8 antibody and F10G6 antibody decreases, but the combined antibody can block the 5'-3' polymerization of Pfu DNA polymerase at 70℃ The enzyme activity and blocking efficiency are both above 97%, which shows that R9C8 antibody and F10G6 antibody play a synergistic role.

Example 5

PCR detection sample amplification effect

Synthesize primer pCDNA3.1-F, with the sequence CTAGAGAACCCACTGCTTAC (SEQ ID NO:22), and primer pCDNA3.1-R, with the sequence TAGAAGGCACAGTCGAGG (SEQ ID NO:23), and dilute to 10 μmol/L.

The sample is the vector pCDNA3.1 plasmid (purchased from Biowind) and added to the negative serum diluted 50 times.

Prepare 10×Pfu buffer 2: 200mmol/L Tris-HCl, 100mmol/L (NH ₄ )·SO ₄ , 100mmol/LKCl, 1% (volume ratio) Tritonx-100, 1mg/mL BSA, 20mmol/L MgSO ₄ , pH8.7(25℃).

The experiment was divided into four groups, namely: antibody group enzyme group, R9C8 antibody enzyme group, F10G6 antibody enzyme group, and Pfu enzyme group.

Antibody enzyme: Take the antibody combination with a concentration of 5 mg/mL, mix it with Pfu DNA polymerase with a concentration of 0.5 mg/mL at a mass ratio of 2:1, and incubate it at 37°C for 30 minutes to form an antibody enzyme.

R9C8 antibody enzyme group: Take the R9C8 antibody with a concentration of 5 mg/mL, mix it with Pfu DNA polymerase with a concentration of 0.5 mg/mL at a mass ratio of 2:1, and incubate it at 37°C for 30 minutes, which is an antibody enzyme.

F10G6 antibody enzyme group: Take F10G6 antibody with a concentration of 5 mg/mL, mix it with Pfu DNA polymerase with a concentration of 0.5 mg/mL at a mass ratio of 2:1, and incubate it at 37°C for 30 minutes to form an antibody enzyme.

Pfu enzyme group: Take another Pfu DNA polymerase with a concentration of 0.5 mg/mL and add the same volume of antibody storage buffer, mix evenly, and incubate at 37°C for 30 minutes.

Prepare according to the formula in Table 2 below

Table 2

Set the temperature program on the PCR machine as shown in Table 3:

table 3

Weigh 1.0g agar powder and add 100mL TAE to prepare a 1.0% electrophoresis gel. Take the above PCR tube, add 6 μL of Loadingbuffer, mix and centrifuge, and load 20 μL onto the electrophoresis gel. Electrophorese at 120V for 30 minutes, observe the electrophoresis bands and take pictures.

The results are shown in Figure 4. The abzyme PCR effect of blocking Pfu DNA polymerase with a combination of antibodies is significantly better than that of Pfu enzyme without antibody blocking and Pfu DNA polymerase with a single antibody (R9C8 abzyme or F10G6 abzyme). The strips are brighter and produce more products.

In summary, the present invention discloses a combination of Pfu DNA polymerase antibodies that can specifically bind to Pfu DNA polymerase and can completely block its 5'-3' polymerase activity at 70°C. The combined antibody and Pfu DNA polymerase The mixed antibody group enzyme is significantly better than Pfu DNA polymerase without antibody and Pfu DNA polymerase with single antibody when amplifying samples.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A Pfu DNA polymerase antibody combination comprising Pfu DNA polymerase monoclonal antibody R9C8 and Pfu DNA polymerase monoclonal antibody F10G6; the sequence of the heavy chain variable region of the monoclonal antibody R9C8 is shown as SEQ ID NO. 7, and the sequence of the light chain variable region of the monoclonal antibody R9C8 is shown as SEQ ID NO. 8; the sequence of the heavy chain variable region of the monoclonal antibody F10G6 is shown as SEQ ID NO. 17, and the sequence of the light chain variable region is shown as SEQ ID NO. 18.

2. The Pfu DNA polymerase antibody combination of claim 1, wherein the CDR1 sequence of the heavy chain variable region sequence of monoclonal antibody R9C8 is shown in SEQ ID No. 1, CDR2 sequence is shown in SEQ ID No. 2, and CDR3 sequence is shown in SEQ ID No. 3.

3. The Pfu DNA polymerase antibody combination of claim 1, wherein the CDR1 sequence of the light chain variable region sequence of monoclonal antibody R9C8 is shown in SEQ ID No. 4, CDR2 sequence is shown in SEQ ID No. 5, and CDR3 sequence is shown in SEQ ID No. 6.

4. The Pfu DNA polymerase antibody combination of claim 1, wherein the CDR1 sequence of the heavy chain variable region sequence of monoclonal antibody F10G6 is shown in SEQ ID No. 11, CDR2 sequence is shown in SEQ ID No. 12, and CDR3 sequence is shown in SEQ ID No. 13.

5. The Pfu DNA polymerase antibody combination of claim 1, wherein the CDR1 sequence of the light chain variable region sequence of monoclonal antibody F10G6 is shown in SEQ ID No. 14, CDR2 sequence is shown in SEQ ID No. 15, and CDR3 sequence is shown in SEQ ID No. 16.

6. The Pfu DNA polymerase antibody combination of claim 1, wherein the heavy chain sequence of the monoclonal antibody R9C8 is shown in SEQ ID NO. 9 and the light chain sequence is shown in SEQ ID NO. 10.

7. Pfu DNA polymerase antibody combination of claim 2, wherein the heavy chain sequence of the monoclonal antibody F10G6 is shown in SEQ ID NO. 19 and the light chain sequence is shown in SEQ ID NO. 20.

8. The Pfu DNA polymerase antibody combination of claim 1, wherein the mass ratio of Pfu DNA polymerase monoclonal antibody R9C8 and Pfu DNA polymerase monoclonal antibody F10G6 is 0.5-1.5:1.

9. use of a Pfu DNA polymerase antibody combination of any one of claims 1 to 8 for reducing non-specific amplification of Pfu DNA polymerase.

10. The use according to claim 9, wherein the mass ratio of Pfu DNA polymerase antibody combination to Pfu DNA polymerase is 1.5-2.5:1.