CN117777298A - Monoclonal antibody combination for blocking Pfu DNA polymerase mutant polymerization activity and application thereof - Google Patents
Monoclonal antibody combination for blocking Pfu DNA polymerase mutant polymerization activity and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody combination for blocking Pfu DNA polymerase mutant polymerization activity and application thereof. The invention firstly discloses a monoclonal antibody combination for blocking the polymerization activity of Pfu DNA polymerase mutant, comprising monoclonal antibody 1H3 and/or monoclonal antibody 6B8. Further disclosed is the use of the above monoclonal antibody combination for reducing non-specific amplification of Pfu DNA polymerase mutants and/or increasing the yield of PCR products. In the monoclonal antibody combination, the monoclonal antibody 1H3 and the monoclonal antibody 6B8 can be respectively and specifically combined with the Thumb structural domain and the Palm structural domain of the Pfu DNA polymerase mutant to seal the polymerization activity of the Pfu DNA polymerase mutant at low temperature or normal temperature, and the specificity of the Pfu DNA polymerase mutant and the yield of long and complex genome PCR can be improved by matching with an optimized reaction solution.
Description
Technical Field
The present invention relates to the field of biotechnology. More particularly, it relates to a monoclonal antibody combination for blocking Pfu DNA polymerase mutant polymerization activity and application thereof.
Background
Polymerase chain reaction (polymerase chain reaction, PCR) technology has been widely used in molecular biology experiments. The PCR technology uses DNA as a template, uses small-fragment single-stranded DNA as a primer, and synthesizes dNTPs into a new DNA strand under the action of DNA polymerase. Thus DNA polymerase is critical to achieving DNA replication in vitro.
Pfu DNA polymerase (also known as Pfu polymerase, or Pfu enzyme) is one of the most commonly used group B polymerases, and Pfu DNA polymerase is a highly thermostable DNA polymerase derived from isolation in thermophilic archaebacteria (Pyrococcus furiosus, pfu). It plays an important role in DNA replication in organisms, and has a DNA polymerase activity at the 5'-3' end and a proofreading activity at the 3'-5' end, both of which are two relatively independent domains (polymerase domain and exonuclease domain) of Pfu enzyme, respectively, acting. Wherein the polymerase domain is further divided into Thumb domain and Palm domain, is the catalytic activity of Pfu enzymeHeart, and key sites for binding to DNA substrates. Because of the Pfu enzyme exonuclease domain, the mutation rate of the bases during DNA replication is very low, namely 1.3X10 －6 (Pavlov A R,Pavlova N V,Kozyavkin S A,et al.Recent developments in the optimization of thermostable DNA polymerases for efficient applications[J]Trends in biotechnology.2004,22 (5): 253-260.). And the half-life of the enzyme is more than 3 hours at 97.5 ℃, so Pfu DNA polymerase with high fidelity and high thermal stability is favored by researchers compared with other DNA polymerases. However, these thermostable DNA polymerases still have a certain polymerase activity under normal or low temperature conditions, and when the 3' -end of the primer is paired with a few bases of a template DNA sequence of a non-amplified target region, non-specific amplification and primer dimer are easily generated.
At present, hot start is a commonly adopted method for solving the problem. The currently commercially available hot start polymerases are hot started by various modifications to the polymerase. The common modification modes are as follows: antibody modification (Kellogg DE, rybalkin I, chen S, et al taq start Antibody: "Hot start" PCR facilitated by a neutralizing monoclonal Antibody directed against Taq DNA polymerase [ J ]. Biotechniques.1994,16 (6): 1134-7.), chemical modification (Moretti T, koons B, budowle B.enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase [ J ]. Biotechniques.1998,25 (4): 716-22.), aptamer (Kainz P, schmiedec-hner A, strack HB. Specificity-enhanced hot-start PCR: addition of double-stranded DNA fragments adapted to the annealing temperature [ J ]. Biotechniques.2000,28 (2): 278-82.), recombinant modification (Kermekchiev MB, tzekov A, barnes WM. Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR [ J ]. Nucleic Acids Res.2003,31 (21): 6139-47.), and some specific nanomaterial modifications (Li H, huang J, 2005-38, lv.38:35, U.J. 35, 35:38). The chemical modification method has the defect of poor repeatability and controllability of an inhibition effect experiment; nucleic acid aptamer methods, recombinant modification methods, and some special nanomaterial modifications are often difficult and costly. Specific antibodies inhibit polymerase activity at low temperatures, reduce non-specific amplification of the reaction, and facilitate long-term thermostability of the polymerase, and thus antibody modification has been widely used in hot start reactions of Taq DNA polymerase (Sharkey DJ, scale ER, christy KG Jr. Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction [ J ]. Biotechnology 1994,12 (5): 506-509.).
Currently, for blocking antibodies for Pfu DNA polymerase, only individual reports are directed against the 3'-5' exonuclease domain (CN 111533806 a) and nanobody (CN 116444675A). The polymerase domain of Pfu DNA polymerase is blocked, so that the Pfu DNA polymerase is not combined with template DNA of a non-amplified target region at low temperature, the amplification of non-specific products is effectively reduced, and the yield of specific products is increased, which is not reported.
Thus, for Pfu DNA polymerase having a polymerase domain mutation, it is highly desired to develop an antibody capable of specifically blocking the polymerase domain activity thereof, thereby further improving the whole Pfu DNA polymerase holoenzyme performance.
Disclosure of Invention
The invention aims to provide a monoclonal antibody combination for blocking the polymerization activity of Pfu DNA polymerase mutant, so as to solve the problems of poor specificity, low yield of target gene products and the like of the existing Pfu DNA polymerase mutant when a long and complex genome template is amplified.
It is another object of the present invention to provide the use of the above monoclonal antibody combination for reducing non-specific amplification of Pfu DNA polymerase and/or increasing yield of PCR products.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly provides a monoclonal antibody combination for blocking Pfu DNA polymerase mutant polymerization activity, wherein the monoclonal antibody combination comprises monoclonal antibody 1H3 and/or monoclonal antibody 6B8;
the monoclonal antibody 1H3 comprises a heavy chain variable region and a light chain variable region, and complementarity determining domains CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO.1 26-33, SEQ ID NO.1 51-58 and SEQ ID NO.1 97-109; the complementarity determining domains CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 36 th positions of SEQ ID NO.2, 54 th to 56 th positions of SEQ ID NO.2 and 93 th to 101 th positions of SEQ ID NO. 2;
the monoclonal antibody 6B8 comprises a heavy chain variable region and a light chain variable region, and complementarity determining domains CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO.3 positions 26-33, SEQ ID NO.3 positions 51-58 and SEQ ID NO.3 positions 97-103; CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.4 at positions 27-32, SEQ ID NO.4 at positions 50-52 and SEQ ID NO.4 at positions 89-97, respectively.
Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H3 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6B8 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
The invention further provides the use of the above monoclonal antibody combination or a monoclonal antibody combination having at least 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 in any of the following:
1) Use in blocking Pfu DNA polymerase mutant polymerization activity;
2) Use in reducing non-specific amplification of Pfu DNA polymerase mutants;
3) The application in improving the yield of PCR products.
In the invention, when a ELISA method is used for specific antibody screening, three antigens, namely Pfu DNA polymerase mutant holoenzyme, pfu DNA polymerase mutant truncated (delta T truncated) deleted by Thumb domain and Pfu DNA polymerase mutant truncated (delta P truncated) deleted by Palm domain, are selected for simultaneous cross screening, and antibodies which can bind Pfu polymerase mutant holoenzyme but not delta T truncated or Pfu DNA polymerase mutant holoenzyme but not delta P truncated are selected. Two monoclonal antibodies are finally screened by the method, and the Thumb structural domain and the Palm structural domain of the Pfu DNA polymerase mutant can be respectively combined through verification, and can be paired to combine with the Pfu DNA polymerase mutant at the same time, and the polymerization activity of the two monoclonal antibodies is blocked at low temperature or normal temperature.
The invention also provides a reaction system which comprises the monoclonal antibody combination and Pfu DNA polymerase mutant.
Further, the molar ratio of monoclonal antibody 1H3, monoclonal antibody 6B8 and Pfu DNA polymerase mutant in the reaction system is 4:1, 2:1, 1:1 and 2:1, 1:1, 0.5:1; preferably, 4:1, 2:1 and 1:1, 0.5:1; more preferably, it is 2:1 and 1:1.
Further, the reaction system also comprises a reaction liquid; the final concentration of each substance in the reaction solution in the reaction system is 80mM Tris-HCl and 40mM K 2 SO 4 、10mM(NH 4 ) 2 SO 4 、0.18mg/mL BSA、10％(v/v)Glycerin、3mM MgSO 4 0.4mM dNTPs; preferably, the pH of the Tris-HCl is 9.0.
The invention further provides application of the reaction system in DNA amplification.
In the present invention, the Pfu DNA polymerase mutant is a Pfu DNA polymerase mutant having Thumb domain and Palm domain mutations. In a specific embodiment of the present invention, the sequence of the Pfu DNA polymerase mutant is shown as SEQ ID NO. 5.
The beneficial effects of the invention are as follows:
the monoclonal antibody 1H3 and the monoclonal antibody 6B8 in the monoclonal antibody combination for blocking the polymerization activity of the Pfu DNA polymerase mutant can be respectively and specifically combined with the Thumb structural domain and the Palm structural domain of the Pfu DNA polymerase mutant to block the polymerization activity of the Pfu DNA polymerase mutant at low temperature or normal temperature, the specificity and the blocking effect of the Pfu DNA polymerase mutant are superior to those of the wild Pfu DNA polymerase, and the specificity of the Pfu DNA polymerase mutant and the yield of long complex genome PCR can be improved by matching with an optimized reaction solution, so that the monoclonal antibody can be widely applied to scientific research, medical diagnosis or other application in the technical means.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 is a graph showing ELISA detection results of 17 hybridoma cell supernatants binding to Thumb domains.
FIG. 2 is a graph showing ELISA detection results of the binding of the supernatant of 14 hybridoma cells to Palm domain.
FIG. 3 is a SDS-PAGE chart showing the results of purification of antibodies produced by 31 hybridoma cells; wherein, 1:PFU-03-101; PFU-03-201; PFU-03-301; PFU-03-401; PFU-03-501; PFU-03-701;7 PFU-03-801; PFU-03-2001; PFU-03-2501; PFU-03-2503; PFU-03-2601; PFU-03-2801; PFU-03-3001; PFU-03-3101; PFU-03-3201;16 PFU-03-3401;17 PFU-03-3501;18 PFU-04-101;19 PFU-04-201;20 PFU-04-501; PFU-04-701;22 PFU-04-801;23 PFU-04-1101;24 PFU-04-2201;25 PFU-04-2701; PFU-04-2801; PFU-04-3001;28 PFU-04-3501;29 PFU-04-3601; PFU-04-3801; PFU-04-4501.
FIG. 4A is a melting curve of Pfu DNA polymerase mutants blocked by 4 antibodies, respectively.
FIG. 4B is a melting curve of Pfu DNA polymerase mutants blocked by 5 antibodies, respectively.
FIG. 5 shows the PCR results of genomic template amplification after blocking Pfu DNA polymerase mutants with 6 antibodies, respectively.
FIG. 6 is a graph showing the results of the double antibody sandwich ELISA method for determining the binding of two monoclonal antibodies 1H3 and 6B8 to Pfu DNA polymerase mutant.
FIG. 7 is a graph showing the results of ELISA method for detecting the affinity of two monoclonal antibodies 1H3 and 6B8 for wild-type Pfu DNA polymerase and Pfu DNA polymerase mutant.
FIG. 8 is a graph showing the effect of different molar ratios of two monoclonal antibody antibodies 1H3 and 6B8 to Pfu DNA polymerase mutant and the combination of the reaction solutions on the amplification ability of Pfu DNA polymerase mutant; wherein A is reaction solution A; b is reaction liquid B.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
EXAMPLE 1 monoclonal antibody acquisition
1. Animal immunization, fusion and primary screening process
1. Immunization of animals
A BALB/C female mouse with the age of 6 weeks is adopted, pfu DNA polymerase mutant holoenzyme (the sequence is shown as SEQ ID NO.5, and the holoenzyme comprises Thumb structural domain and Palm structural domain), and a conventional immunization method is adopted, wherein the immunization route is subcutaneous injection, and the specific method is as follows:
the Pfu DNA polymerase mutant was mixed with Freund's complete adjuvant in equal volumes at 30. Mu.g and injected subcutaneously at 3 to 4 points on the backs of mice; performing secondary immunization with the same dosage after three weeks; three more weeks later, the same dose immunization was performed for three times; the boost was performed for two more weeks, and each mouse was intraperitoneally injected with 30. Mu.g pfu DNA polymerase mutant and an equal volume of saline mixed to obtain an immunogen.
2. Cell fusion
Cell fusion was performed 3 days after the last immunization.
Preparation of spleen cell suspension: on the day of cell fusion, taking immunized BALB/c mice, removing eyeballs for blood collection, separating mouse serum as a positive control in antibody detection, and taking spleens of the mice to prepare spleen cell suspension;
preparation of myeloma cell suspension: resuscitates cells in advance for two weeks, ensures that the cells are in logarithmic growth phase when in use, and obtains myeloma cell suspension;
preparation of feeder cells: the day before fusion, mouse abdominal macrophages are obtained and cultured in 96-well plates to obtain feeder cells.
Cell fusion: adopting PEG-mediated fusion method, taking spleen cell suspension and myeloma cell suspension according to spleen cellsMixing with myeloma cells at a ratio of 5:1 in serum-free DMEM medium, centrifuging at 1200rpm for 5 min, removing supernatant, flicking the bottom of a centrifuge tube with fingers, loosely mixing the two cells, placing in a beaker containing 37 ℃ water, keeping warm, adding 1mL 50% PEG (pH 8.0) fusion cells within 1 min, shaking while adding, standing for 30 sec after adding, adding serum-free DMEM medium to terminate fusion, centrifuging at 1000rpm for 5 min, suspending the precipitate with HAT medium, and packaging into 96-well cell plates containing feeder cells at 37deg.C and 5% CO 2 Is cultured in a cell culture incubator.
3. Hybridoma cell screening
When the fusion cells cover 10% -50% of the bottom of the wells, three antigens, pfu DNA polymerase mutant holoenzyme, pfu DNA polymerase mutant truncated form deleted Thumb domain (delta T truncated form) and Pfu DNA polymerase mutant truncated form deleted Palm domain (delta P truncated form), are used for screening positive and negative wells by indirect ELISA method, and hybridoma cell culture supernatant is detected. Finally, 36 hybridoma cell strains with strong antigen binding capacity are obtained through screening.
The indirect ELISA method comprises the following specific experimental procedures:
(1) Coating: with coating buffer (Na 2 CO 3 1.59g，NaHCO 3 2.95g, deionized water to 1000 mL) to dilute Pfu DNA polymerase mutant holoenzyme, deltaT truncate and DeltaP truncate to 1. Mu.g/mL, respectively, adding to an ELISA plate, 100. Mu.L/well, and incubating at 37℃for 2 hours;
(2) Washing the plate: plates were washed 1 time with wash solution (PBST: 1 XPBS with 0.05% TWEEN-20), 300. Mu.L/well;
(3) Closing: 150. Mu.L/well 0.5% BSA solution, 37℃for 2 hours;
(4) Adding a sample: the hybridoma cell culture supernatants were added to the coated ELISA plates of (1), 50. Mu.L/well, respectively.
(5) Incubation: 37 ℃ for 1 hour;
(6) Washing the plate: washing the plate 5 times with a washing liquid, 300. Mu.L/well;
(7) Enzyme conjugate: will beGoat Anti-Mouse IgG (H+L), HRP Conjugate, diluted 1:5000 with dilution buffer (1% BSA in PBST), 100. Mu.L/well, incubated at 37℃for 30 min;
(10) Washing the plate: washing the plate 5 times with a washing liquid, 300. Mu.L/well;
(11) A substrate: adding TMB substrate, 100 mu L/hole, and incubating for 15 minutes at 37 ℃ in dark place;
(12) And (3) terminating: 0.5. 0.5M H 2 SO 4 100. Mu.L/well;
(13) And (3) measuring: readings are taken at a wavelength of 450nm dominant wavelength, 620nm reference wavelength.
4. Hybridoma cell verification screening
The 36 hybridoma cell culture supernatants obtained by the above screening were subjected to respective verification of Thumb domain and Palm domain binding.
ELISA plate coating was performed using Thumb domain and Palm domain proteins, respectively, and the hybridoma cell culture supernatants were examined. The results of the indirect ELISA assay (the method is the same as the specific experimental procedure of the indirect ELISA assay described in example 1-hybridoma cell screening) are shown in FIGS. 1 and 2, and 17 hybridoma cells and 14 hybridoma cell culture supernatants can bind to Thumb domain and Palm domain, respectively.
2. Antibody purification
The 31 strains of hybridoma cells are inoculated into mouse ascites for antibody production, protein A filler is used for antibody purification, and the specific operation steps are as follows:
(1) And (3) column loading: 6mL of Protein A packing was loaded into a gravity column and 10 column volumes were washed with deionized water.
(2) Balance: 10 column volumes were equilibrated using 1 XPBS and prepared for loading.
(3) Loading and rebalancing: directly loading by gravity, controlling the flow rate of the ascites sample to be about 1mL/min according to the liquid level height difference, and catching the FT effluent by a clean pipe. After the sample was applied, the sample was further equilibrated with 1 XPBS for about 20 column volumes, FT was temporarily stored at 4℃and FT was sampled.
(4) Eluting: before elution, 25. Mu.L of a neutralization buffer (2M Tris-HCl, water as solvent, pH 8.0) was added to a collection tube in a proportion of 1mL of an eluent (100 mM Glycine, water as solvent, pH 3.2) to obtain an elution buffer, and the elution was performed with the elution buffer, and the eluted fractions were collected in separate tubes, 3mL each tube.
The purified antibody is subjected to SDS-PAGE (12%) electrophoresis detection, and the result is shown in figure 3, so that 31 monoclonal antibodies are finally obtained, the protein purity is high, and the antibody can be used for the next detection.
3. Polymerase blocking active antibody screening
The 31 monoclonal antibodies and Pfu DNA polymerase mutants are incubated according to a molar ratio of 1:1, the antibody blocking enzyme is obtained at 37 ℃ for 1 hour, pfu DNA polymerase mutant naked enzymes (namely Pfu naked enzymes) without antibody blocking are used as a control, and then a qPCR system is configured for melting curve measurement. The measurement method is as follows:
the reaction solution was prepared according to the qPCR reaction System of Table 1, and then was started up, and CFX Opus Real-Time PCR System was used. The qPCR reaction procedure was: the first step is that the reaction is carried out for 30 seconds at 37 ℃ for pre-denaturation; and in the second step, the reaction is carried out for 10 seconds at 37 ℃, the reaction is carried out for 2 minutes at 37 ℃ for 65 times of cyclic reactions, finally, a melting curve is established, and the activity of the antibody-blocked Pfu polymerase is evaluated according to the amplification curve and the melting curve.
TABLE 1 qPCR reaction System
| Component (A) | Volume of | Working concentration |
| 2 XBuffer (enzyme free) | 10μL | 1× |
| 10μM ssDNA | 0.6μL | 0.3μM |
| 10μM Oligo 1C | 0.6μL | 0.3μM |
| 100×SYBR GreenⅠ | 0.18μL | 0.9× |
| H 2 O | 8.02μL | - |
| Pfu naked enzyme/antibody blocking enzyme | 0.6μL | - |
The results showed that 22 antibodies without blocking activity could be eliminated, the remaining 9 antibodies with blocking activity, but the 9 antibodies were different in blocking activity, and 6 monoclonal antibodies with better blocking activity were further obtained according to the dissolution profile (as shown in FIG. 4A and FIG. 4B), PFU-03-201, PFU-03-301, PFU-03-501, PFU-03-3501, PFU-04-101 and PFU-04-201, respectively.
4. Antibody blocking effect verification
And selecting 6 monoclonal antibodies with better blocking activity screened by the melting curve method, designing experiments, performing PCR amplification to verify the blocking effect, and setting 2 compound holes in each group of experiments.
Incubating the 6 monoclonal antibodies and Pfu DNA polymerase mutant according to a molar ratio of 1:1, performing 1h at 37 ℃, taking Pfu DNA polymerase mutant naked enzyme without antibody blocking (namely Pfu naked enzyme) as a control, and then configuring a PCR reaction system to perform PCR reaction to amplify target DNA.
Wherein, the PCR reaction system is as follows:
the procedure for the PCR reaction was as follows:
the PCR amplification results are shown in FIG. 5, and compared with Pfu DNA polymerase mutant naked enzymes (shown as 'Pfu naked enzymes' in the figure) without antibody blocking, pfu DNA polymerase mutants with 6 antibody blocking have certain enhancement on the specificity of genome DNA template amplification, and the quantity of partial antibody blocking enzyme amplification products is improved.
5. Antibody pairing
To screen an antibody combination from the above antibodies, the Thumb domain and Palm domain of Pfu DNA polymerase mutant can be combined at the same time for improvement and optimization of blocking effect.
The antibody is subjected to antibody pairing screening by using a double-antibody sandwich ELISA and a chessboard method, and the specific steps are as follows:
(1) Biotin labeling: all 6 antibodies are respectively labeled with Biotin according to the mol ratio of 1:20 with Biotin, and after being uniformly mixed, the reaction is carried out for 1 hour at room temperature, and after the dialysis at 4 ℃ is finished, the Biotin labeled antibody, namely the Biotin-antibody is obtained;
(2) Coating: diluting all 6 antibodies respectively by using a coating buffer solution, adding an ELISA plate, and incubating for 2 hours at 37 ℃;
(3) Washing the plate: washing the plate 1 time with PBST;
(4) Closing: 5% BSA solution, blocked at 37℃for 2 hours;
(5) Sample adding: diluting Pfu DNA polymerase mutant to corresponding concentration, loading 100 mu L/hole, and incubating for 2 hours at room temperature;
(6) Washing the plate: washing the plate with a washing liquid for 5 times;
(7) Adding a detection antibody: diluting the Biotin-antibody of the step (1) with a diluent buffer solution according to a ratio of 1:1000, and incubating for 1 hour at room temperature after sample addition;
(6) Washing the plate: washing the plate with a washing liquid for 5 times;
(7) Add Avidin-HRP: diluting the Avidin-HRP with Avidin-HRP diluent, wherein the dilution ratio is 1:1000, and 100 mu L/hole is added for incubation for 30 minutes at room temperature;
(8) Washing the plate: washing the plate 5 times with a washing liquid, 300. Mu.L/well; the last time of plate washing is performed, and then the plate is patted dry;
(9) Color development: adding TMB substrate, 100 mu L/hole, and incubating for 15 minutes at 37 ℃ in dark place;
(10) And (3) terminating: 0.5. 0.5M H 2 SO 4 100. Mu.L/well;
(11) And (3) measuring: readings are taken at a wavelength of 450nm dominant wavelength, 620nm reference wavelength.
The detection result shows that when PFU-03-301 is used as a coating antibody and Biotin-PFU-04-201 is used as a detection antibody, the two antibodies can show good pairing linear relationship, and the result is shown in FIG. 6, and PFU DNA polymerase mutant can be identified. As can be seen from the step one, the two antibodies respectively bind to Thumb domain and Palm domain and recognize different epitopes. They were designated as 1H3 and 6B8 antibodies, respectively.
6. Acquisition of hybridoma antibody Gene sequences
Monoclonal antibodies 1H3 and 6B8 were subtype identified using the mouse antibody subtype identification kit. The subtype of monoclonal antibody 1H3 is IgG 1 The subtype of monoclonal antibody 6B8 was IgG 2b The light chain subtypes are all kappa.
Extracting total RNA of hybridoma cells, reversely transcribing the total RNA into cDNA, and respectively amplifying heavy chain variable region sequences and light chain variable region sequences of two antibodies by using nested PCR (polymerase chain reaction) with the cDNA as a template to finally obtain 1H3 and 6B8 heavy chain variable region sequences, wherein the following specific steps are as follows:
the heavy chain variable region amino acid sequence of the monoclonal antibody 1H3 is as follows:
QVQLQQSGGELVKPGASVKMSCKAFGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNDDTKYNEKFKGKAKLTVEKSSSTVYLELSRLTSDDSAVYYCARGYRSDWDYFDYWGQGTTLTVSS
the amino acid sequence of the heavy chain CDR1 is GYTFTTYP
The heavy chain CDR2 amino acid sequence is FHPYNDDT
The heavy chain CDR3 amino acid sequence is ARGYRSDWDYFDY;
the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H3 is shown as SEQ ID NO.1, and complementarity determining domains CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.1 positions 26-33, SEQ ID NO.1 positions 51-58 and SEQ ID NO.1 positions 97-109 respectively.
The amino acid sequence of the light chain variable region of the monoclonal antibody 1H3 is as follows:
DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLYWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPWTFGGGTKLEIK
the amino acid sequence of the light chain CDR1 is KSVSTSGYSY
The amino acid sequence of the light chain CDR2 is LAS
The light chain CDR3 amino acid sequence is QHSRELPWT
The amino acid sequence of the light chain variable region of the monoclonal antibody 1H3 is shown as SEQ ID NO.2, and complementarity determining domains CDR1, CDR2 and CDR3 of the light chain variable region are shown as 27 th to 36 th positions of SEQ ID NO.2, 54 th to 56 th positions of SEQ ID NO.2 and 93 th to 101 th positions of SEQ ID NO.2 respectively.
The heavy chain variable region amino acid sequence of the monoclonal antibody 6B8 is as follows:
QVQLQQPGAELVQPGASVRLSCKASSAFATSYWMHWVKQRPGQGLEWIGEINPNSGRTNYNEKFQSKAALTVDKSSSTAYMQLSSLTSEDSAVYYCASQVRGYWGQGTTLTVSS
the heavy chain CDR1 amino acid sequence is SAFATSYW
Heavy chain CDR2 amino acid sequence INPNSGRT
The amino acid sequence of the heavy chain CDR3 is ASQVRGY
The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6B8 is shown as SEQ ID NO.3, and complementarity determining domains CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.3 positions 26-33, SEQ ID NO.3 positions 51-58 and SEQ ID NO.3 positions 97-103 respectively;
the light chain variable region amino acid sequence of the monoclonal antibody 6B8 is
DIQMTQSPSSLSASLGERVSLTCRASQEISGYLYWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPYTFGGGTKLEIK
The light chain CDR1 amino acid sequence is QEISGY
The amino acid sequence of the light chain CDR2 is AAS
The light chain CDR3 amino acid sequence is LQYASYPYT
The amino acid sequence of the light chain variable region of the monoclonal antibody 6B8 is shown as SEQ ID NO.4, and complementarity determining domains CDR1, CDR2 and CDR3 of the light chain variable region are shown as 27 th to 32 th positions of SEQ ID NO.4, 50 th to 52 th positions of SEQ ID NO.4 and 89 th to 97 th positions of SEQ ID NO.4 respectively.
EXAMPLE 2 verification of binding specificity of monoclonal antibodies 1H3 and 6B8 to Pfu DNA polymerase mutant
To verify whether the two monoclonal antibodies 1H3 and 6B8 screened had selectivity between wild-type Pfu DNA polymerase and Pfu DNA polymerase mutant. The affinity of different concentrations (0, 0.015, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1. Mu.g/mL) of monoclonal antibodies 1H3 and 6B8 for both polymerases (i.e.wild-type Pfu DNA polymerase and Pfu DNA polymerase mutant) was examined using an indirect ELISA method (the method is the same as the specific experimental procedure for the indirect ELISA method described in example 1-hybridoma cell screening).
The ELISA binding curve results are shown in FIG. 7, and show that at the same initial concentration, both monoclonal antibodies 1H3 and 6B8 have strong binding capacity to Pfu DNA polymerase mutant, and that both monoclonal antibodies have strong selectivity and specificity to Pfu DNA polymerase mutant.
EXAMPLE 3 molar ratio of monoclonal antibodies 1H3 and 6B8 to Pfu DNA polymerase mutant and improvement of the amplification ability of Pfu DNA polymerase mutant by ion reaction solution
The molar ratios of monoclonal antibodies 1H3, 6B8 and Pfu DNA polymerase mutants in the PCR reaction system of example 1 were set to 4:1, 2:1, 1:1, 0:1 and 2:1, 1:1, 0.5:1, 0:1, respectively. According to the PCR reaction system of example 1, an ion buffer system was provided to give a reaction solution A (the final concentration of each substance in the reaction solution was 80mM Tris-HCl (pH 9.0), 40mM K) 2 SO 4 、10mM(NH 4 ) 2 SO 4 、0.18mg/mL BSA、10％(v/v)Glycerin、3mM MgSO 4 0.4mM dNTPs) and reaction solution B (final concentration of each of the reaction solution in the reaction system was 80mM Tris-HCl (pH 9.5), 20mM K 2 SO 4 、10mM(NH 4 ) 2 SO 4 、0.18mg/mL BSA、10％(v/v)Glycerin、6mM MgSO 4 0.4mM dNTPs). Experiments were performed with a total of 4×4×2, and 32 different combinations of experimental conditions, and the Pfu DNA polymerase mutants of different reaction systems were tested for their amplification capacity using the procedure of the PCR reaction in example 1.
As a result, as shown in FIG. 8, when the molar ratio of the monoclonal antibodies 1H3, 6B8 and Pfu DNA polymerase mutant (denoted by "Pfu enzyme" in the figure) was 2:1:1, and the ion buffer system was the reaction solution A, the amplified product amount was the highest, and the specificity was the best.
It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.
Claims (10)
1. A monoclonal antibody combination that blocks Pfu DNA polymerase mutant polymerization activity, characterized in that the monoclonal antibody combination comprises monoclonal antibody 1H3 and/or monoclonal antibody 6B8;
the monoclonal antibody 1H3 comprises a heavy chain variable region and a light chain variable region, and complementarity determining domains CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO.1 26-33, SEQ ID NO.1 51-58 and SEQ ID NO.1 97-109; the complementarity determining domains CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as 27 th to 36 th positions of SEQ ID NO.2, 54 th to 56 th positions of SEQ ID NO.2 and 93 th to 101 th positions of SEQ ID NO. 2;
the monoclonal antibody 6B8 comprises a heavy chain variable region and a light chain variable region, and complementarity determining domains CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO.3 positions 26-33, SEQ ID NO.3 positions 51-58 and SEQ ID NO.3 positions 97-103; CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID NO.4 at positions 27-32, SEQ ID NO.4 at positions 50-52 and SEQ ID NO.4 at positions 89-97, respectively.
2. The monoclonal antibody combination according to claim 1, wherein the amino acid sequence of the heavy chain variable region of monoclonal antibody 1H3 is shown in SEQ ID No.1 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6B8 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.
3. Use of a monoclonal antibody combination according to claim 1 or 2 or a monoclonal antibody combination having at least 80%, 85%, 90%, 95%, 98% or 99% or more identity with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 for blocking the polymerization activity of Pfu DNA polymerase mutants.
4. Use of a monoclonal antibody combination according to claim 1 or 2 or a monoclonal antibody combination having at least 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 for reducing the non-specific amplification of Pfu DNA polymerase mutants.
5. Use of a monoclonal antibody combination according to claim 1 or 2 or a monoclonal antibody combination having at least 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 for increasing the yield of PCR products.
6. A reaction system comprising the monoclonal antibody combination of claim 1 or 2 and Pfu DNA polymerase mutant.
7. The reaction system of claim 6, wherein the molar ratio of monoclonal antibody 1H3, monoclonal antibody 6B8 and Pfu DNA polymerase mutant in the reaction system is 4:1, 2:1, 1:1 and 2:1, 1:1, 0.5:1; preferably, 4:1, 2:1 and 1:1, 0.5:1; more preferably, it is 2:1 and 1:1.
8. The reaction system of claim 6, wherein the reaction system further comprises a reaction liquid; the final concentration of each substance in the reaction solution in the reaction system is 80mM Tris-HCl and 40mM K 2 SO 4 、10mM(NH 4 ) 2 SO 4 、0.18mg/mL BSA、10％Glycerin、3mM MgSO 4 0.4mM dNTPs; preferably, the pH of the Tris-HCl is 9.0.
9. Use of a reaction system according to any one of claims 6-8 for DNA amplification.
10. The monoclonal antibody combination according to claim 1 or 2, or the use according to any one of claims 3-5 and 9, or the reaction system according to any one of claims 6-8, characterized in that the Pfu DNA polymerase mutant is a Pfu DNA polymerase mutant having a Thumb domain and a Palm domain;
preferably, the Pfu DNA polymerase mutant has a sequence shown in SEQ ID NO. 5.
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