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Monoclonal antibody specifically combined with Taq DNA polymerase and application thereof

A monoclonal antibody and polymerase technology, applied in the field of antibodies, can solve the problems of difficulty in fully activating the activity, difficult to achieve hot start, difficult to block the activity, etc.

Active Publication Date: 2022-07-01
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the chemical modification method is more commonly used, the activity is more completely sealed, and no exogenous pollution will be introduced, but its activity is difficult to fully activate, and it needs to be incubated at high temperature for a long time
However, the activity of the aptamer method is difficult to completely seal, and the temperature often rises above 45°C to restore the activity, and it is difficult to achieve a strict hot start.

Method used

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  • Monoclonal antibody specifically combined with Taq DNA polymerase and application thereof
  • Monoclonal antibody specifically combined with Taq DNA polymerase and application thereof
  • Monoclonal antibody specifically combined with Taq DNA polymerase and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0078] The preparation method of the hybridoma cell of the present invention is as follows: firstly, the mice are immunized with Taq DNA polymerase, and the mice whose serum titer and neutralization activity meet the requirements are screened. Then, the spleen cells of the screened mice that meet the requirements are fused with myeloma cells such as SP2 / 0Ag14 myeloma cells, and after effective dilution, cloning, HAT screening, immunogen detection, etc. and Taq DNA polymerase active hybridoma cells.

[0079] Of course, in addition to the myeloma cell SP2 / 0Ag14 myeloma cell, which is particularly suitable for the present invention, any other myeloma cell that can be used to prepare hybridoma cells can be used, and all fall within the scope of the present invention.

[0080] In the third aspect of the present invention, there is provided a polynucleotide sequence encoding the monoclonal antibody of the first aspect of the present invention, the polynucleotide sequence comprising:...

Embodiment 1

[0125] Example 1 Selection of high-affinity and neutralizing mice

[0126] The mice were immunized three times with Taq DNA polymerase as the antigen, and the serum titers of the immunized mice were detected. Specifically, the recombinant Taq DNA polymerase (SEQ ID NO: 29, purity of more than 90%, protein stored in 1×PBS buffer) derived from Thermus Aquaticus was used as the antigen, and 5 BALB / c mice were subjected to immunization experiments in a conventional manner, and the immunization experiments were performed three times.

[0127] After immunization, mouse serum titer ELISA was performed to confirm the affinity activity of the antibodies produced after immunization. In the ELISA test, the immunogenic protein, namely recombinant Taq DNA polymerase, was used to coat, and the serum titer of at least one animal reached 1:128000. The steps of ELISA detection are as follows: (1) Using Taq DNA polymerase as the antigen, dilute it to an appropriate concentration (0.5-10 μg / m...

Embodiment 2

[0152] Example 2. Cell fusion and screening of hybridoma cell lines

[0153] Mice E were sacrificed and fixed on a dissection plate. The spleen was taken out by cutting off the connective tissue with a knife and placed in a glass homogenizer for homogenization. The obtained spleen cells were diluted to a certain concentration, and then mixed with SP2 / 0Ag14 myeloma. Cells were subjected to cell fusion assays. Fusion cells were limited dilution, cloned in 96-well plates, and hybridomas were screened by HAT.

[0154] The cell culture conditions are as follows: a) The cell laboratory is routinely sterilized and irradiated with ultraviolet light for 30 minutes. b) The culture medium was placed in a constant temperature water bath at 37°C for more than 20min. c) Take out the cryovial from the liquid nitrogen tank, immediately put it into a 37°C water bath, and shake it quickly until the cryopreservation solution is completely thawed. d) Transfer the cell suspension into a 15ml ce...

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Abstract

The invention relates to the field of antibodies, in particular to a monoclonal antibody specifically combined with Taq DNA polymerase and application of the monoclonal antibody. The monoclonal antibody provided by the invention is specifically combined with Taq DNA polymerase to neutralize the excision and / or polymerization activity of the Taq DNA polymerase, and releases the combined Taq DNA polymerase under a thermal activation condition, so that the excision and / or polymerization activity of the Taq DNA polymerase is recovered.

Description

technical field [0001] The present invention relates to the field of antibodies. More specifically, the present invention relates to monoclonal antibodies that specifically bind to Taq DNA polymerase and uses thereof. Background technique [0002] PCR technology involving Taq DNA polymerase is widely used in biochemical tests and in vitro diagnostic industries. Taq enzyme is a high temperature enzyme, the enzyme still has a certain activity at room temperature. Therefore, before PCR predenaturation, the 5'-3' exonucleation activity of Taq DNA polymerase can cause degradation of primers, probes or templates, while its polymerization activity can cause template or primer mismatches before PCR predenaturation. Non-specific amplification; in the subsequent PCR cycle, non-specific products can be further amplified, resulting in a decrease in the yield of the target product and even failure to amplify the target band. [0003] The hot-start method can effectively solve the abov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13C12N5/20C12N15/10C12N9/12C12R1/91
CPCC07K16/40C12N9/1252C12Q1/686C12Y207/07007C07K2317/56C07K2317/565C07K2317/76C12Q2521/101
Inventor 高重亮陈茁谢庆庆郑越董宇亮章文蔚
Owner SHENZHEN HUADA GENE INST
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