CN116731184A - Monoclonal antibody F6H12 specifically binding to Taq enzyme and application thereof - Google Patents
Monoclonal antibody F6H12 specifically binding to Taq enzyme and application thereof Download PDFInfo
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Abstract
The invention provides a monoclonal antibody F6H12 specifically combined with Taq enzyme, wherein the CDR1 sequence of the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 1, the CDR2 sequence is the amino acid sequence shown in SEQ ID NO. 2, and the CDR3 sequence is the amino acid sequence shown in SEQ ID NO. 3; the CDR1 sequence of the light chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 4, the CDR2 sequence is the amino acid sequence shown in SEQ ID NO. 5, and the CDR3 sequence is the amino acid sequence shown in SEQ ID NO. 6. The monoclonal antibody F6H12 has continuous and stable blocking polymerase effect at different temperatures.
Description
Technical Field
The invention relates to a monoclonal antibody F6H12 specifically binding to Taq enzyme and application thereof.
Background
Polymerase chain reaction (PCR: polymerase ChainReaction) is a technique for exponentially amplifying a specific nucleotide fragment in vitro by repeated cycles of heat denaturation of a double-stranded DNA template, primer annealing, and primer extension using two short single-stranded primers at both ends of the DNA fragment, using a thermostable DNA polymerase. Taq enzyme is DNA polymerase isolated from Thermus aquaticus YT1 castor strain. Taq enzyme plays a role in PCR reaction, namely, DNA is used as a template by utilizing the activity of 3 '. Fwdarw.5' polymerase, and deoxymononucleotide in dNTPs is added to the end of 3-OH one by one; meanwhile, the 5 '. Fwdarw.3 ' exonuclease activity can be used for identifying and eliminating mismatched primer ends, which are related to the correction function in the replication process, can hydrolyze nucleotides from the 5' ends, and can also act through a plurality of nucleotides to cut mismatched nucleotides.
The Taq enzyme antibody is an anti-Taq enzyme antibody for hot start PCR, which inhibits DNA polymerase activity after binding to Taq enzyme. In PCR amplification, the Taq enzyme antibody and Taq enzyme are combined to inhibit the activity of DNA polymerase before high temperature denaturation, so that the non-specific annealing of the primer and the non-specific amplification caused by the primer dimer can be effectively inhibited under the low temperature condition. The Taq enzyme antibody is denatured in the initial DNA denaturation step of the PCR reaction, and the activity of the DNA polymerase is recovered, so that the hot start PCR effect is achieved.
However, at present, the existing Taq enzyme antibody has a stable blocking effect on the activity of polymerase at low temperature and normal temperature, and when the temperature is further increased, the blocking effect of the polymerase is drastically reduced, and it is difficult to maintain a continuous stable blocking effect, thereby leading to an increase of nonspecific amplification. Thus, there is a need in the art for a Taq enzyme antibody that is capable of maintaining a sustained and stable polymerase blocking effect.
Disclosure of Invention
The invention provides a monoclonal antibody F6H12 specifically combined with Taq enzyme and application thereof, which can effectively solve the problems.
The invention is realized in the following way:
a monoclonal antibody F6H12 specifically binding to Taq enzyme has a CDR1 sequence of a heavy chain variable region sequence shown in SEQ ID NO. 1, a CDR2 sequence of an amino acid sequence shown in SEQ ID NO. 2 and a CDR3 sequence of an amino acid sequence shown in SEQ ID NO. 3; the CDR1 sequence of the light chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 4, the CDR2 sequence is the amino acid sequence shown in SEQ ID NO. 5, and the CDR3 sequence is the amino acid sequence shown in SEQ ID NO. 6.
As a further improvement, the heavy chain variable region sequence of the monoclonal antibody F6H12 which specifically binds to Taq enzyme is the amino acid sequence shown in SEQ ID NO. 7.
As a further improvement, the light chain variable region sequence of the monoclonal antibody F6H12 which specifically binds to Taq enzyme is the amino acid sequence shown in SEQ ID NO. 8.
As a further improvement, the heavy chain sequence of the monoclonal antibody F6H12 which specifically binds to Taq enzyme is the amino acid sequence shown as SEQ ID NO. 9.
As a further improvement, the light chain sequence of the monoclonal antibody F6H12 which specifically binds to Taq enzyme is the amino acid sequence shown as SEQ ID NO. 10.
The application of the monoclonal antibody F6H12 in the construction of the non-specific amplification of the hot start Taq enzyme for reducing the Taq enzyme.
A cell producing the monoclonal antibody F6H12.
The beneficial effects of the invention are as follows: the monoclonal antibody F6H12 provided by the invention can keep continuous and stable polymerization activity of sealing Taq DNA polymerase 5'-3' in a temperature range of 0-65 ℃, and the sealing efficiency is more than 97%, so that the non-specific amplification and primer dimer generation can be effectively reduced. As the temperature increases to 75 ℃, monoclonal antibody F6H12 begins to dissociate from Taq enzyme, restoring Taq enzyme polymerization activity. Compared with the non-blocked Taq enzyme, the specific amplification efficiency is obviously improved.
The abzyme can well amplify samples with low concentration of 15 copies/reaction, and the amplification sensitivity of the abzyme is obviously improved compared with that of wild Taq enzyme. The amplification CT value and the commercial antibody enzyme ratio are improved to a certain extent, and the fluorescence value is higher, so that the amplification sensitivity is better.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of a Taq enzyme antibody polymerase activity blocking assay.
FIG. 2 shows the blocking detection result of the Taq enzyme exonuclease activity.
FIG. 3 is a graph showing the amplification effect of the Taq enzyme antibody provided in example 4 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
Example 1
Preparation of monoclonal antibody specifically binding Taq enzyme
1. Immunization of animals
Taking 8-12 week old BALB/c mice, fully and uniformly mixing 100 mug/mouse recombinant antigen containing wild Taq enzyme protein with equivalent Freund's complete adjuvant, injecting into the abdominal cavity of the mice, fully and uniformly mixing 100 mug/mouse recombinant antigen containing wild Taq enzyme protein with equivalent Freund's incomplete adjuvant every 2 weeks, and injecting into the abdominal cavity of the mice for multiple times to strengthen immunity. The serum (indirect ELISA) titer of the detected mice is more than 1:5000, and the mice can be used for fusion, and the mice are boosted in abdominal cavity for 3 days before fusion, and the dosage is 50 mug/mouse.
2. Preparation of feeder cells
BALB/c murine peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mice were killed by pulling the neck, soaked in 75% alcohol whole body, placed in a super clean bench, cut off the abdominal skin with scissors under aseptic operation, expose the peritoneum, pour in 5mL of RPMI1640 basal culture solution with syringe abdominal cavity, repeatedly wash, recover the washing liquid, centrifuge at 1000rpm for 5 minutes, leave sediment, re-suspend in complete culture solution with RPMI1640 containing HAT, and adjust the cell concentration to 1×10 5 Each mL was incubated overnight at 37℃in 96-well plates, 150. Mu.L/well and 5% CO 2.
3. Preparation of immune spleen cells
Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a plate, rinsed once with RPMI1640 basal medium, placed on a nylon mesh of a small beaker, and ground and filtered to prepare a cell suspension. Centrifugation, discarding supernatant, re-suspending the RPMI1640 basal medium, repeating the above steps three times, and counting.
4. Cell fusion
(1) Taking 40mL of HAT culture solution, 15mL of DMEM serum-free culture solution and 1mL of 50% PEG (M12000), and respectively placing in a water bath at 37 ℃ for pre-heating;
(2) Separate mouse myeloma cells Sp2/0 (2-5×10) 7 Individual), the above-mentioned immune spleen cells (10) 8 And b) adding the suspension into a 50mL centrifuge tube, uniformly mixing, and adding DMEM serum-free culture solution to 40mL. Centrifuging for 10 min, pouring out supernatant, and mixingHomogenizing;
(3) The centrifuge tube was placed in 37℃pre-warmed water, 0.7mL of pre-warmed 50% PEG solution was taken and allowed to stand for 90 seconds. Immediately dropwise adding 15mL of preheated serum-free culture solution at 37 ℃;
(4) The DMEM serum-free medium was added to 40mL, centrifuged for 10 minutes, and the supernatant was discarded. 40mL of HAT culture solution containing 15% -20% of fetal bovine serum is added. Mixing with a pipette, dripping into small holes of 4 96-well cell culture plates containing feeder cells, and culturing in an incubator at 37 ℃ and 7% CO2 at 2 drops per well.
5. Selective culture of hybridoma cells
The cells were cultured with the HAT medium described above at days 1,3,5 and 7 after cell fusion, and true hybrid cells were selected.
6. Detection of specific antibodies and hybridoma cell cloning
And (3) absorbing the supernatant of each culture hole, detecting the culture holes containing the antibody specifically recognizing the Taq enzyme protein recombinant antigen in the culture solution by using an indirect ELISA method, and screening to obtain the antibody F6H12 with better reactivity.
The amino acid sequence of the Taq enzyme monoclonal antibody F6H12 is as follows:
heavy chain CDR1 sequence: YWMH (SEQ ID NO: 1)
Heavy chain CDR2 sequence: MAPSNETGLQNFSD (SEQ ID NO: 2)
Heavy chain CDR3 sequence: GLRLRYFDY (SEQ ID NO: 3)
Light chain CDR1 sequence: ASQDSNYL (SEQ ID NO: 4)
Light chain CDR2 sequence: YSRLH (SEQ ID NO: 5)
Light chain CDR3 sequence: QQNTLPT (SEQ ID NO: 6)
The heavy chain variable region sequences are:
EVQLQQSGPELVRPGASVKMSCKASGYTFIYWMHWVKQRPGQGL EWIGMAPSNETGLQNFSDKATLSVDKSSNTAYIQFSSLTSEDSAVYFCAR GLRLRYFDYWGQGTTLTVSS(SEQ ID NO:7)
the light chain variable region sequences are:
DIVMTQTTSSLSVSLGDRVTISCASQDSNYLWYQQKPDGTVKLLIY YSRLHGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQNTLPTFGGGTKL EIK(SEQ ID NO:8)
the heavy chain sequence of the antibody is as follows:
EVQLQQSGPELVRPGASVKMSCKASGYTFIYWMHWVKQRPGQGLEWIGMAPSNETGLQNFSDKATLSVDKSSNTAYIQFSSLTSEDSAVYFCARGLRLRYFDYWGQGTTLTVSSSTPPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:9)
the antibody light chain sequence is:
DIVMTQTTSSLSVSLGDRVTISCASQDSNYLWYQQKPDGTVKLLIYYSRLHGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQNTLPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:10)
the heavy and light chain sequences (SEQ ID NOS: 9 and 10) of the above antibodies were synthesized by Shanghai Biotechnology (Shanghai) Inc., and inserted into pcDNA3.1 vector. According to FreeStyle of Thermo Fisher Co TM 293 Expression System User Manual the vector containing the heavy and light chain sequences was transfected into 293-F cells, cultured and the cell culture supernatant collected.
Cell culture supernatants were isolated and purified by protein A affinity chromatography, operating according to the protocol of Cytiva company handbook, affinity Chromatography, vol 1: antibodies.
Purifying to prepare the Taq enzyme monoclonal antibody F6H12.
The above procedures are well known to those skilled in the art, and other plasmid construction, cell transfection, culture methods, and isolation and purification techniques in the known art may be used to obtain the monoclonal antibodies.
Example 2
Polymerase activity blocking detection of monoclonal antibody specifically binding Taq enzyme
And uniformly mixing the Taq enzyme antibody F6H12 and wild Taq enzyme according to the mass ratio of 2:1 to prepare the abzyme.
Taking hairpin oligonucleotide probe with sequence of
5'-TAGCGAAGGATGTGAACCTAATCCCTGCTCCCGCGGCCGATCTGC CGGCCGCGG-3' (SEQ ID NO: 11), diluted to 100. Mu. Mol/L.
Preparing 10 XPCR buffer:250mmol/L Tris-HCl,50mmol/L (NH) 4 )·SO 4 500mmol/L KCl,1% (volume ratio) Triton X-100, pH8.8 (25 ℃ C.), 25mmol/L MgCl 2 ，25mmol/L dNTP。
Formulated according to the formulation shown in table 1, all operations were performed on ice with three replicates for each experiment:
TABLE 1
Different temperature programs are set on a fluorescent quantitative PCR instrument, the amplification temperature is 37 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃,75 ℃ and the amplification time is 16s, and the sealing effect at different temperatures is detected.
PCR was performed for 120 cycles.
Fluorescence acquisition channel: FAM (Fam)
The PCR octant pipe added with the reaction liquid is placed in front of a fluorescent quantitative PCR instrument, and the fluorescent quantitative PCR instrument is preheated for half an hour according to the temperature, and the polymerization reaction is started.
After the reaction, the difference between the fluorescence value of the 30 th cycle and the initial value was calculated from the fluorescence quantitative PCR data. The 5'-3' polymerase activity blocking effect of the test enzyme = 100% to the difference in front-to-back fluorescence of the test enzyme/the difference in front-to-back fluorescence of the wild-type enzyme.
As shown in FIG. 1, according to the graph, the blocking efficiency can reach more than 97% by blocking the Taq DNA polymerase 5'-3' continuously and stably in the temperature range of 0-65 ℃ through blocking the monoclonal antibody F6H12. At 75 ℃, the antibody is significantly inactivated and the polymerase activity begins to release.
Example 3
Monoclonal antibody exonuclease activity blocking detection for specific binding Taq enzyme
And uniformly mixing the Taq enzyme antibody F6H12 and the Taq enzyme according to the mass ratio of 2:1 to prepare the antibody-Taq enzyme.
Hairpin oligonucleotide probe with sequence of
5-FAM-CTAGCTC(BHQ)CATGGTCCGTAGGCGTAACGGTCCCGGGT AGATCCCGGTCCGATGGGCCTTAGACTGTC-3’
(SEQ ID NO: 12), diluted to 100. Mu. Mol/L.
Preparing 10 XPCR buffer:250mmol/L Tris-HCl,50mmol/L (NH 4). SO 4 500mmol/L KCl,1% (volume ratio) Triton X-100, pH8.8 (25 ℃ C.), 25mmol/L MgCl 2 ，25mmol/L dNTP。
Formulated according to the following table 2 formulation, all operations were performed on ice, and each experiment was repeated in triplicate:
TABLE 2
| Reaction liquid component | Dosage (mu L) |
| 10×PCR buffer | 2.5 |
| Hairpin oligonucleotide probe | 0.05 |
| Wild Taq enzyme or antibody enzyme | 0.5 |
| Purified water | 21.95 |
| Totals to | 25 |
Setting different temperature programs on a fluorescence quantitative PCR instrument, wherein the amplification temperature is 37 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃,75 ℃ and the amplification time is 30s;80 cycles; fluorescence acquisition channel: FAM. And detecting the sealing effect at different temperatures.
The PCR eight-joint tube added with the reaction liquid is placed in front of a fluorescent quantitative PCR instrument, and the fluorescent quantitative PCR instrument is preheated for half an hour according to the temperature, and the reaction is started.
After the reaction, the difference between the fluorescence value of the 80 th cycle and the initial value was calculated from the fluorescence quantitative PCR data. The 5'-3' exonuclease activity blocking effect of the test enzyme = 100% -the difference in front-to-back fluorescence of the test enzyme/the difference in front-to-back fluorescence of the wild-type enzyme.
The results are shown in FIG. 2. As can be seen from FIG. 2, antibody F6H12 has a constant exonuclease blocking capacity at each temperature, which is only 20% at most. The reason for this analysis is thought to be that steric hindrance of the antibody inhibits the exonuclease activity of the Taq enzyme antibody.
Example 4
q pcr detection of HBV sample amplification effect
Synthetic primer HBV-L with sequence CAATCACTCACCAACCTCCTG
(SEQ ID NO: 13), primer HBV-R, sequence CGGGCAACATACCTTGATAA (SEQ ID NO: 14), probe HBV-P, sequence 5'-FAM-CCAATTTGTCCTGGTTATCG (BHQ) -3' (SEQ ID NO: 15).
Diluted to 100. Mu. Mol/L.
The sample is HBV positive serum, serum/plasma free DNA extraction kit (product number DP 339) purchased from Tiangen biochemical technology is adopted to extract sample DNA, and the copy number of the sample DNA is measured according to the q-pcr kit of HBV, so that a 15copies/5 mu L low-concentration sample is prepared.
Preparing 10 XPCR buffer 2:250mmol/L Tris-HCl,50mmol/L (NH) 4 )·SO 4 500mmol/L KCl,1% (volume ratio) Triton X-100, pH8.8 (25 ℃ C.), 25mmol/L MgCl 2 ，25mmol/L dNTP，1mmol/L EDTA·2Na。
The experiment is divided into three groups, namely an antibody enzyme group, a wild Taq enzyme group and a commercial antibody enzyme group.
Abzyme: taking antibody F6H12 with the concentration of 5mg/mL, and respectively mixing the antibody F6H12 with wild Taq DNA polymerase with the concentration of 0.5mg/mL according to the following ratio of 2:1, and incubating for 30min at 37 ℃ to obtain the abzyme.
Wild Taq enzyme: the wild Taq DNA polymerase with the concentration of 0.5mg/mL is added with the antibody storage buffer solution with the same volume, and the mixture is evenly mixed and incubated for 30min at 37 ℃.
Commercial enzyme: the antibody (purchased from TAKARA) was taken to be mixed with wild Taq DNA polymerase at a concentration of 0.5mg/mL uniformly in a mixing ratio of 2:1.
formulated according to the following Table 3 formulation
TABLE 3 Table 3
The temperature program set on the fluorescent quantitative PCR instrument is shown in table 4:
TABLE 4 Table 4
As shown in FIG. 3, the antibody enzyme can amplify the sample with low concentration of 15 copies/reaction, and compared with the wild Taq enzyme, the sensitivity of the antibody enzyme is greatly improved. Compared with the commercial enzyme, the antibody enzyme amplification CT value is improved to a certain extent, and the fluorescence value is higher.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A monoclonal antibody F6H12 specifically binding to Taq enzyme is characterized in that the CDR1 sequence of the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 1, the CDR2 sequence is the amino acid sequence shown in SEQ ID NO. 2, and the CDR3 sequence is the amino acid sequence shown in SEQ ID NO. 3; the CDR1 sequence of the light chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 4, the CDR2 sequence is the amino acid sequence shown in SEQ ID NO. 5, and the CDR3 sequence is the amino acid sequence shown in SEQ ID NO. 6.
2. The monoclonal antibody F6H12 which specifically binds to Taq enzyme according to claim 1, wherein the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 7.
3. The monoclonal antibody F6H12 which specifically binds to Taq enzyme according to claim 1, wherein the light chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 8.
4. The monoclonal antibody F6H12 which specifically binds to Taq enzyme according to claim 1, wherein the heavy chain sequence is the amino acid sequence shown in SEQ ID NO. 9.
5. The monoclonal antibody F6H12 which specifically binds to Taq enzyme according to claim 1, wherein the light chain sequence is the amino acid sequence shown in SEQ ID NO. 10.
6. Use of the monoclonal antibody F6H12 of any one of claims 1 to 5 for constructing a non-specific amplification of a hot start Taq enzyme to reduce Taq enzyme.
7. A cell producing the monoclonal antibody F6H12 of any one of claims 1 to 5.
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