Pfu DNA polymerase antibody combination and application thereof

Abstract

The application provides a Pfu DNA polymerase antibody combination, which comprises 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 as SEQ ID NO:1, and the light chain variable region sequence is shown as SEQ ID NO:2; the heavy chain variable region sequence of the monoclonal antibody F10G6 is shown as SEQ ID NO:3, and the light chain variable region sequence is shown as SEQ ID NO:4. The combination has better 5'-3' end polymerase activity of Pfu DNA enzyme in a wide temperature range (0-70 DEG C) before the pre-denaturation stage.

Description

TECHNICAL FIELD

[0001] The present application relates to a Pfu DNA polymerase antibody combination and its application, belonging to the technical field of Pfu DNA polymerase. BACKGROUND

[0002] Polymerase chain reaction (PCR) is a molecular biology technology for amplifying specific DNA fragments. It has the characteristics of strong specificity, high sensitivity, rapidity, simplicity and good repeatability, and 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 mother strand DNA as a template and primers as a starting point through denaturation, annealing and extension cycles, thereby achieving DNA replication.

[0003] Pfu DNA polymerase (Pfu enzyme for short) is a DNA polymerase isolated from Pyrococcus furiosus (Pfu) by scientists. The enzyme contains two protein subunits (P45 and P50), is a polymer, and has a molecular weight of 90 kda, with excellent thermal stability. Unlike Taq DNA polymerase, the enzyme has both 5'-3' polymerase activity and 3'-5' exonuclease activity, and the main function of 3'-5' exonuclease activity is correction, which can immediately recognize and remove mismatched nucleotides, making Pfu enzyme have extremely high fidelity. Due to high fidelity and high stability, Pfu enzyme has become one of the most widely used DNA polymerases.

[0004] In the process of PCR, Pfu enzyme, like other DNA polymerases, still has certain polymerase activity under low temperature conditions, thereby producing non-specific amplification and forming primer dimers. At present, many ways can be used to achieve the purpose of hot start, such as gene mutation of the enzyme, physical isolation, chemical modification, nucleic acid primer modification, etc. However, these methods have some disadvantages, such as incomplete enzyme activity blocking, affecting the complexity of PCR, affecting the continuity of the enzyme, affecting the fidelity or the method being relatively cumbersome, etc.

[0005] In view of the above-mentioned related art, a relatively better method is to modify Pfu enzyme with monoclonal antibodies. Pfu enzyme antibody is a hot start PCR anti-Pfu enzyme antibody, which inhibits the activity of DNA polymerase after binding with Pfu enzyme. During PCR amplification, the Pfu enzyme antibody binds with Pfu enzyme to inhibit the activity of DNA polymerase before high-temperature denaturation, which can effectively inhibit the non-specific annealing of primers and the non-specific amplification caused by primer dimers under low-temperature conditions, and improve the specificity of amplification and the yield of target DNA. However, in the existing PCR, only one Pfu DNA polymerase antibody is usually used. If the temperature is above 70℃, the blocking efficiency of the Pfu DNA polymerase antibody to the Pfu DNA polymerase is greatly reduced, resulting in some non-specific amplification. SUMMARY

[0006] The present application provides a Pfu DNA polymerase antibody combination and its application, which can effectively solve the above-mentioned problems.

[0007] The present application is implemented as follows:

[0008] A Pfu DNA polymerase antibody combination, comprising 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, and 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.

[0009] In some embodiments, the CDR1 sequence of the heavy chain variable region sequence of the monoclonal antibody R9C8 is shown in SEQ ID NO: 1, the CDR2 sequence is shown in SEQ ID NO: 2, and the CDR3 sequence is shown in SEQ ID NO: 3,

[0010] In some embodiments, the CDR1 sequence of the light chain variable region sequence of the monoclonal antibody R9C8 is shown in SEQ ID NO: 4, the CDR2 sequence is shown in SEQ ID NO: 5, and the CDR3 sequence is shown in SEQ ID NO: 6.

[0011] In some embodiments, the CDR1 sequence of the heavy chain variable region sequence of the monoclonal antibody F10G6 is shown in SEQ ID NO: 11, the CDR2 sequence is shown in SEQ ID NO: 12, and the CDR3 sequence is shown in SEQ ID NO: 13.

[0012] In some embodiments, the CDR1 sequence of the light chain variable region sequence of the monoclonal antibody F10G6 is shown as SEQ ID NO: 14, the CDR2 sequence is shown as SEQ ID NO: 15, and the CDR3 sequence is shown as SEQ ID NO: 16.

[0013] In some embodiments, the monoclonal antibody R9C8 has a heavy chain sequence shown as SEQ ID NO: 9 and a light chain sequence shown as SEQ ID NO: 10.

[0014] In some embodiments, the monoclonal antibody F10G6 has a heavy chain sequence shown as SEQ ID NO: 19 and a light chain sequence shown as SEQ ID NO: 20.

[0015] 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.

[0016] A Pfu DNA polymerase antibody combination as described above is used to reduce the non-specific amplification of Pfu DNA polymerase.

[0017] In some embodiments, the mass ratio of the Pfu DNA polymerase antibody combination and the Pfu DNA polymerase is 1.5-2.5:1.

[0018] The present application has the following beneficial effects:

[0019] The present application provides a Pfu DNA polymerase antibody combination, which includes the R9C8 antibody and the F10G6 antibody, and the two antibodies have a synergistic effect. The combination has a better 5'-3' polymerase activity blocking effect on the Pfu DNA polymerase in a wide temperature range (0-70℃) before the denaturation stage. The blocking efficiency of the Pfu DNA polymerase 5'-3' polymerase activity at 70℃ is more than 97%. The antibody combination greatly reduces the non-specific amplification in PCR. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0021] Figure 1 is a graph of the polymerase activity blocking efficiency of the Pfu enzyme antibody combination.

[0022] Figure 2 is the blocking efficiency graph of the polymerase activity of the Pfu enzyme R9C8 antibody.

[0023] Figure 3 is the blocking efficiency graph of the polymerase activity of the Pfu enzyme F10G6 antibody.

[0024] Figure 4 is the nucleic acid electrophoresis graph of the PCR results of the antibody group enzyme, the single antibody enzyme (R9C8 antibody enzyme and F10G6 antibody enzyme) and the Pfu DNA polymerase. DETAILED DESCRIPTION

[0025] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0026] In the description of the present application, the terms "first", "second" are only used for description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "multiple" is two or more, unless otherwise explicitly and specifically limited.

[0027] Example 1

[0028] Preparation of Pfu DNA polymerase

[0029] 1. Acquisition, synthesis and vector construction of the gene of Pfu DNA polymerase

[0030] According to the sequence of Pfu DNA polymerase found by NCBI (NCBI: NC_003413.1), an Nde I restriction site is introduced at the N-terminal of the gene, a stop codon and a Xho I restriction site are introduced at the C-terminal of the gene, and the gene is synthesized (Shanghai Sunway Biotech). The gene and the pET28a vector are double digested by Nde I and Xho I restriction enzymes, respectively, and then T4 DNA ligase is used for construction to the pET28a-Pfu vector.

[0031] 2. Construction and expression of recombinant Pfu DNA polymerase bacteria

[0032] The constructed pET28a-Pfu vector was transformed into E. coli BL21 (DE3) strain (purchased from Shanghai Shengong Bioengineering) and plated on LB plates, incubated overnight, and single colonies were picked. Inoculate 10 mL of LB liquid medium, add Kan antibiotic to a final concentration of 1 mM, incubate at 37°C, 250 rpm overnight, the next day transfer to 200 mL of LB medium at a ratio of 1:100, add Kan antibiotic to a final concentration of 1 mM, incubate at 37°C, 250 rpm until the OD value is 0.6-0.8, add isopropyl thiogalactoside to a final concentration of 0.01 mM / L-0.2 mM / L, and induce expression at 25°C for 6-8 h. Centrifuge the bacterial suspension to collect the bacterial cells, centrifuge at 4°C, 8000 rpm for 10 min.

[0033] 3. Purification of Pfu DNA polymerase

[0034] (1) Mix according to the mass ratio of bacterial cells to Buffer A (Tirs-HCl 20 mM, sodium chloride 500 mM, imidazole, 10 mM, adjust pH to 7.4) 1:10, ice bath ultrasonic disruption, disruption conditions: power 300 W, ultrasonic 3 S, pause 8 S, disruption 15 min;

[0035] (2) Centrifuge at 4°C, 12000 rpm for 30 min, collect the supernatant, filter the supernatant with a 0.22 μm filter membrane, and load onto a Ni chromatography column;

[0036] (3) Wash the impurities with Buffer B (Tirs-HCl 20 mM, sodium chloride 500 mM, imidazole, 20 mM, adjust pH to 7.4);

[0037] (4) Elute Pfu DNA polymerase with Buffer C (Tirs-HCl 20 mM, sodium chloride 500 mM, imidazole, 200 mM, DTT 8 mM, adjust pH to 7.4);

[0038] (5) Ultrafiltration and concentration of the purified Pfu DNA polymerase, and store in storage Buffer (Tris-HCl (pH 7.4) 20 mM, KCl 100 mM, DTT 1 mM, 0.5% Tween 20, 0.1 mM EDTA, glycerol 50% (v / v).

[0039] Example 2

[0040] Preparation of monoclonal antibodies

[0041] Two monoclonal antibodies of Pfu DNA polymerase are prepared in the application, which are R9C8 and F10G6 respectively.

[0042] The antibody is prepared as follows:

[0043] The reagent or kit involved in the application and its source are as follows:

[0044] Freund's Adjuvant, Complete (item number 77140, Thermo Fisher); Freund's Adjuvant, Incomplete (item number 77145, Thermo Fisher); HAT Media Supplement (50x) (item number: 21060017, Thermo Fisher); HT Media Supplement (50x) (item number H0111067030, Thermo Fisher); PEG (item number P7181, sigma company); RPMI 1640 (item number L210KJ, Shanghai Yuanpei Biological); FBS (C04001-500, Shanghai Xiaopeng Biological); DMEM (item number L310KJ, Shanghai Yuanpei Biological); Penicillin-St reptomycin (item number 15140122, gibco); HRP labeled goat anti-mouse antibody (item number D110087, Shanghai Shengong Biological); Protein A Resin (item number SA023010, Changzhou Tian Dili and Man Biological Technology Co., Ltd.).

[0045] 1. Mouse immunization:

[0046] 8-10 weeks old, about 20g, healthy Balb / c female mice are immunized with Pfu DNA polymerase. The first immunization is subcutaneous injection of 50ug of immunogen obtained by mixing Pfu DNA polymerase with Freund's complete adjuvant in equal volume on the back of the mouse. Two weeks later, the second immunization is subcutaneous injection of 50ug of immunogen obtained by mixing Pfu DNA polymerase with Freund's incomplete adjuvant in equal volume on the back of each mouse. Two weeks later, the third immunization is subcutaneous injection of 50ug of immunogen obtained by mixing Pfu DNA polymerase with Freund's incomplete adjuvant in equal volume on the back of each mouse. Two weeks later, the booster immunization is intraperitoneal injection of 50ug of immunogen obtained by mixing Pfu DNA polymerase with physiological saline in equal volume in each mouse.

[0047] 2. Cell Fusion: This process begins three days after the last immunization of mice. BALB / c mice were collected three days after the last immunization. Blood was collected by enucleation, and the mouse serum was separated as a positive control for antibody detection. Simultaneously, mice were euthanized by cervical dislocation, and their spleens were harvested to prepare a spleen cell suspension. Myeloma cells were resuscitated two weeks in advance (ensuring the cells were in the logarithmic growth phase at the time of use). One day before fusion, mouse peritoneal macrophages were obtained and cultured in 96-well plates to obtain cell plates containing feeder cells. Using the PEG-mediated fusion method, spleen cell suspension and myeloma cell suspension were mixed at a cell ratio of 5:1 in serum-free DMEM medium, centrifuged at 1200 rpm for 5 min, the supernatant was removed, and the bottom of the centrifuge tube was gently tapped with a finger to loosely mix the two types of cells. The tube was then incubated in a beaker containing 37°C water. Within 1 minute, 1 mL of 50% PEG (pH 8.0) fusion cell solution was added while shaking. After the addition was complete, the tube was allowed to stand for 30 seconds, and then serum-free DMEM medium was added to terminate the fusion. The tube was centrifuged at 1000 rpm for 5 min, the pellet was resuspended in HAT medium, and aliquoted into 96-well cell culture plates containing feeder cells. Cells were cultured in a 37°C, 5% CO2 incubator. The preparation of 500 mL of HAT medium required 100 mL FBS, 5 mL penicillin-streptomycin, 10 mL HAT media supplement, and 385 mL DMEM.

[0048] 3. Hybridoma cell screening: After culturing in a cell culture incubator for 7 days, when the fusion cells cover 10%-50% of the bottom of the wells, positive wells are screened using a conventional indirect ELISA method. The hybridoma cell culture supernatant is plated with Pfu DNA polymerase and tested. The secondary antibody is enzyme-labeled goat anti-mouse antibody. Mouse serum separated in step 2 is used as a positive control. Positive hybridoma cells with high antibody titers are selected.

[0049] 4. Hybridoma cell cloning: The day before cloning, prepare feeder cells and plate them according to step 2; 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 per well; culture at 37°C and 5% CO2 for 7-10 days, and detect antibodies when visible clones appear; observe under an inverted microscope and mark the wells with only a single clone growing, and finally obtain the monoclonal antibody hybridoma cell line through preliminary screening.

[0050] 5. Preparation of monoclonal antibody ascites: 0.5 ml of liquid paraffin was injected intraperitoneally into Balb / c mice. Ten days later, the selected hybridoma cell line was inoculated with 1×10⁻⁶ cells. 6The ascites were extracted from the abdominal cavity of the Balb / c mice. After about 10 days, the abdomen of the mice began to swell, and the mice were sacrificed by cervical dislocation, and the ascites were extracted once.

[0051] 6. Purification of the monoclonal antibody: centrifugation at 3000 rpm for 10 min, and the colorless and transparent middle layer was the ascites. The ascites and saturated (NH4)2SO4 were mixed at a ratio of 1:1, and then centrifuged at 10000 rpm for 10 min. The supernatant was discarded, and the precipitate was redissolved in a PB solution with a pH of 7.0 and a concentration of 20 mM. The solution was passed through a protein A column, and then eluted with a Gly-HCl solution with a pH of 2.7 and a concentration of 100 mM. The eluate was immediately neutralized with a Tris-HCl solution with a pH of 9.0 and a concentration of 1 M. The subsequent PBS solution was replaced with a PBS solution at 4°C after 6 h of dialysis, and the purified monoclonal antibodies R9C8 and F10G6 of Pfu DNA polymerase were obtained.

[0052] The sequencing results are as follows:

[0053] The amino acid sequence of R9C8 is as follows:

[0054] The heavy chain CDR1 sequence is YCRT (SEQ ID NO: 1)

[0055] The heavy chain CDR2 sequence is QGHDNETDKPRILS (SEQ ID NO: 2)

[0056] The heavy chain CDR3 sequence is GLSNKIHDL (SEQ ID NO: 3)

[0057] The light chain CDR1 sequence is QSLYANDS (SEQ ID NO: 4)

[0058] The light chain CDR2 sequence is YSTLY (SEQ ID NO: 5)

[0059] The light chain CDR3 sequence is QQWTLPT (SEQ ID NO: 6)

[0060] The heavy chain variable region sequence is: QVQLQQSGAELVKPGTSVKLSCKASGYNFI YCRTWVKLRPGQGLEWIGQGHDNETDKPRILSKATLTVDKSSSTAYMQL SGLASADSAVYYCTR GLSNKIHDLWGQGTTLTVSS (SEQ ID NO: 7) The light chain variable region sequence is:

[0061] DVVMTQSTPSLSVSLGDRVTISCQSLYANDSWYQQKPGTVPKLLIY QSLYANDSRVPSRFSASGSGTDFSLTISNLEQEDFATYFCYSTLYFGGGTK LEIK (SEQ ID NO: 8)

[0062] The heavy chain sequence is:

[0063] QVQLQQSGAELVKPGTSVKLSCKASGYNFIYCRTWVKLRPGQGLEWIGQGHDNETDKPRILSKATLTVDKSSSTAYMQLSGLASADSAVYYCTRGLSNKIHDLWGQGTTLTVSSSTPPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 9)

[0064] The light chain sequence is:

[0065] DVVMTQSTPSLSVSLGDRVTISCQSLYANDSWYQQKPGTVPKLLIY QSLYANDSRVPSRFSASGSGTDFSLTISNLEQEDFATYFCYSTLYFGGGTK LEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 10)

[0066] The amino acid sequence of F10G6 is as follows:

[0067] Heavy chain CDR1 sequence: YTMH (SEQ ID NO: 11)

[0068] Heavy chain CDR2 sequence: WAPSINTNYEKFKD (SEQ ID NO: 12)

[0069] Heavy chain CDR3 sequence: HGRKTFDY (SEQ ID NO: 13)

[0070] Light chain CDR1 sequence: ASQTDSYL (SEQ ID NO: 14)

[0071] Light chain CDR2 sequence: YSYLE (SEQ ID NO: 15)

[0072] Light chain CDR3 sequence: QQKENPT (SEQ ID NO: 16)

[0073] The heavy chain variable region sequence is: QVQLQQSGAELVKPGASVKLSCKMSAYNFTYTMHWVKQRPGQGLEWIGWAPSINTNYEKFKDKATLSVDTSSNTAYMQLSSLTSEDSALYYCARHGRKTFDYWGQGTTLTVSA (SEQ ID NO: 17) and the light chain variable region sequence is:

[0074] DIVMTQTTASLAVSLGDRATISCASQTDSYLWYQQKPGQPPKLLIKYSYLEGVPSRFSGSGSGTDYSLTIHPVEQEDIATYYCQQKENPTFGGGTKLEIK (SEQ ID NO: 18)

[0075] The heavy chain sequence is:

[0076] QVQLQQSGAELVKPGASVKLSCKMSAYNFTYTMHWVKQRPGQGLEWIGWAPSINTNYEKFKDKATLSVDTSSNTAYMQLSSLTSEDSALYYCARHGRKTFDYWGQGTTLTVSASTPPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 19)

[0077] The light chain sequence is:

[0078] DIVMTQTTASLAVSLGDRATISCASQTDSYLWYQQKPGQPPKLLIKYSYLEGVPSRFSGSGSGTDYSLTIHPVEQEDIATYYCQQKENPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 20)

[0079] The antibody combination was mixed with F10G6 at a mass ratio of 1:1.

[0080] Example 3

[0081] Preparation of antibody enzyme and Pfu enzyme

[0082] The antibody combination was mixed with Pfu DNA polymerase at a mass ratio of 2:1, and the concentration of the antibody combination was 5 mg / mL and the concentration of the Pfu DNA polymerase was 0.5 mg / mL. The mixture was incubated at 37°C for 30 min, and the antibody enzyme was obtained.

[0083] Take antibody R9C8, concentration is 5 mg / mL, respectively, with the concentration of 0.5 mg / mL Pfu DNA polymerase according to 2:1 mass ratio mixed evenly, 37℃ incubation for 30 min, that is R9C8 antibody enzyme.

[0084] Take antibody F10G6, concentration is 5 mg / mL, respectively, with the concentration of 0.5 mg / mL Pfu DNA polymerase according to 2:1 mass ratio mixed evenly, 37℃ incubation for 30 min, that is F10G6 antibody enzyme.

[0085] Take another concentration of 0.5 mg / mL Pfu DNA polymerase mixed with the same volume of antibody storage buffer evenly, 37℃ incubation for 30 min, as Pfu enzyme.

[0086] Example 4

[0087] 5'-3' polymerase activity detection

[0088] Take hairpin type oligonucleotide

[0089] TAGCGAAGGATGTGAACCTAATCCCTGCTCCCGCGGCCGATCTGCCG GCCGCGG (SEQ ID NO: 21), diluted to 100 μmol / L.

[0090] Prepare 10×Pfu buffer: 250 mmol / L Tris-HCl, 50 mmol / L (NH4)·SO4, 500 mmol / L KCl, 1% (volume ratio) Triton x-100, pH 8.8 (25℃), 25 mmol / L MgCl2, 25 mmol / L dNTP.

[0091] According to the following table 1 formula preparation, all operations are carried out on ice, each experiment has three repeats:

[0092] Table 1

[0093]

[0094]

[0095] Set the temperature program on the fluorescence quantitative PCR instrument:

[0096] The amplification temperature is 37℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃; The amplification time is 16 s; 120 cycles; Fluorescence collection channel; FAM.

[0097] The fluorescence quantitative PCR instrument was preheated for half an hour at the above temperature, and the PCR eight-tube tube containing the reaction liquid was placed in the fluorescence quantitative PCR instrument to start the polymerization reaction.

[0098] After the reaction, the difference between the fluorescence value at the 120th cycle and the initial value was calculated according to the fluorescence quantitative PCR data. The blocking effect of the 5'-3' polymerase activity of the enzyme was detected = 1- the difference in fluorescence before and after the test antibody enzyme / the difference in fluorescence before and after the Pfu enzyme.

[0099] The results are shown in Figures 1-3 R9C8 antibody can block the 5'-3' polymerase activity of Pfu DNA polymerase at 65°C, and the blocking efficiency is more than 95%; F10G6 antibody can block the 5'-3' polymerase activity of Pfu DNA polymerase at 60°C, and the blocking efficiency is more than 98%; at 70°C, the blocking efficiency of R9C8 antibody and F10G6 antibody decreases, but the combined antibody can block the 5'-3' polymerase activity of Pfu DNA polymerase at 70°C, and the blocking efficiency is more than 97%, which shows that R9C8 antibody and F10G6 antibody have a synergistic effect.

[0100] Example 5

[0101] PCR detection sample amplification effect

[0102] Synthetic primer pCDNA3.1-F, sequence is CTAGAGAACCCACTGCTTAC (SEQ ID NO: 22), primer pCDNA3.1-R, sequence is TAGAAGGCACAGTCGAGG (SEQ ID NO: 23), diluted to 10 μmol / L.

[0103] The sample is the vector pCDNA3.1 plasmid (purchased from Bio Wind) added to 50 times diluted negative serum.

[0104] Prepare 10×Pfu buffer 2: 200 mmol / L Tris-HCl, 100 mmol / L (NH4)·SO4, 100 mmol / L KCl, 1% (volume ratio) Triton x-100, 1 mg / mL BSA, 20 mmol / L MgSO4, pH 8.7 (25°C).

[0105] The experiment is divided into four groups, respectively: antibody group, enzyme group, R9C8 antibody enzyme group, F10G6 antibody enzyme group, Pfu enzyme.

[0106] Antibody enzyme group: Take antibody combination, concentration is 5 mg / mL, respectively with concentration 0.5 mg / mL Pfu DNA polymerase is mixed uniformly according to 2:1 mass ratio, 37 DEG C incubation 30 min, it is antibody enzyme.

[0107] R9C8 antibody enzyme group: take R9C8 antibody, concentration is 5 mg / mL, respectively with concentration 0.5 mg / mL Pfu DNA polymerase is mixed uniformly according to 2:1 mass ratio, 37 DEG C incubation 30 min, it is antibody enzyme.

[0108] F10G6 antibody enzyme group: take F10G6 antibody, concentration is 5 mg / mL, respectively with concentration 0.5 mg / mL Pfu DNA polymerase is mixed uniformly according to 2:1 mass ratio, 37 DEG C incubation 30 min, it is antibody enzyme.

[0109] Pfu enzyme group: another concentration 0.5 mg / mL Pfu DNA polymerase is mixed uniformly with the same volume of antibody storage buffer, 37 DEG C incubation 30 min.

[0110] According to the following table 2 formula preparation

[0111] Table 2

[0112]

[0113]

[0114] The temperature program is set on the PCR instrument as shown in table 3:

[0115] Table 3

[0116]

[0117] Take 1.0 g agar powder into 100 mL TAE to prepare 1.0% electrophoresis gel. Take the above PCR tube, add 6 μL of Loading buffer, mix and centrifuge 20 μL sample to electrophoresis gel. Electrophoresis under 120 V for 30 min, observe the electrophoresis band and take a photo.

[0118] The results are shown in Figure 4 The antibody enzyme PCR effect of blocking Pfu DNA polymerase with antibody combination is obviously better than that of Pfu enzyme without antibody blocking and Pfu DNA polymerase with single antibody (R9C8 antibody enzyme or F10G6 antibody enzyme), and the amplification band is brighter and the product is more.

[0119] The application discloses a Pfu DNA polymerase antibody combination which can specifically bind to Pfu DNA polymerase and completely block 5'-3' polymerase activity at 70 DEG C.

[0120] The above merely describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Pfu DNA polymerase antibody combination, characterized in that, The invention includes 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, and 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.

2. The Pfu DNA polymerase antibody combination according to claim 1, characterized in that, The heavy chain variable region sequence of the monoclonal antibody R9C8 is shown in SEQ ID NO:1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3.

3. The Pfu DNA polymerase antibody combination according to claim 1, characterized in that, The CDR1 sequence of the light chain variable region of the monoclonal antibody R9C8 is shown in SEQ ID NO:4, the CDR2 sequence is shown in SEQ ID NO:5, and the CDR3 sequence is shown in SEQ ID NO:

6.

4. The Pfu DNA polymerase antibody combination according to claim 1, characterized in that, The heavy chain variable region sequence of the monoclonal antibody F10G6 is shown in SEQ ID NO:11 for CDR1, SEQ ID NO:12 for CDR2, and SEQ ID NO:13 for CDR3.

5. The Pfu DNA polymerase antibody combination according to claim 1, characterized in that, The CDR1 sequence of the light chain variable region of the monoclonal antibody F10G6 is shown in SEQ ID NO:14, the CDR2 sequence is shown in SEQ ID NO:15, and the CDR3 sequence is shown in SEQ ID NO:

16.

6. The Pfu DNA polymerase antibody combination according to claim 1, characterized in that, 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. The Pfu DNA polymerase antibody combination according to claim 2, characterized in that, 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 according to claim 1, characterized in that, The mass ratio of the Pfu DNA polymerase monoclonal antibody R9C8 to the Pfu DNA polymerase monoclonal antibody F10G6 is 0.5-1.5:

1.

9. The use of the Pfu DNA polymerase antibody combination according to any one of claims 1 to 8 in reducing the non-specific amplification of Pfu DNA polymerase.

10. The application according to claim 9, characterized in that, The mass ratio of the Pfu DNA polymerase antibody combination to Pfu DNA polymerase is 1.5-2.5:1.

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