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Taq DNA polymerase monoclonal antibody combination as well as reaction system and application thereof

A monoclonal antibody and reaction system technology, applied in the direction of enzymes, transferases, enzyme stabilization, etc., can solve the problems of long-term stability of enzymes, product reduction, and amplification capacity decline.

Active Publication Date: 2020-10-23
BEIJING TRANSGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, due to the time-consuming and labor-intensive antibody screening, it is difficult to screen for the best antibody. Although non-specific amplification can be reduced to a certain extent, the increase in the antibody in the system will cause the decrease in polymerase activity and the long-term stability of the enzyme. Affected, the overall amplification ability is reduced, the product is reduced, etc.

Method used

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  • Taq DNA polymerase monoclonal antibody combination as well as reaction system and application thereof
  • Taq DNA polymerase monoclonal antibody combination as well as reaction system and application thereof
  • Taq DNA polymerase monoclonal antibody combination as well as reaction system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Animal immunization, fusion and preliminary screening process

[0074] (1) Animal immunization: 8-week-old BALB / C female mice were used, and the antigen was EasyTaq DNA polymerase, and the conventional immunization method was used for immunization. The specific immunization method:

[0075] For the first immunization, the immunogen obtained by subcutaneously injecting 50 μg of EasyTaq DNA polymerase and Freund’s complete adjuvant in equal volumes was injected subcutaneously at 3 to 4 points on the back of each mouse;

[0076] Two weeks later, the second immunization was carried out, and the immunogen obtained by subcutaneously injecting 50 μg of EasyTaq DNA polymerase and Freund’s incomplete adjuvant in equal volumes was injected subcutaneously at 3 to 4 points on the back of each mouse;

[0077] Three immunizations were carried out two weeks later, and the immunogen obtained by mixing equal volumes of 50 μg EasyTaq DNA polymerase and Freund's incomplete adjuv...

Embodiment 2

[0091] Embodiment 2 Expansion inhibition screening of hybridoma cells

[0092] In order to detect whether the antibody secreted by the initially screened hybridoma cell line has the effect of blocking the active region of EasyTaq DNA polymerase, the molecular beacon detection experiment is used for verification. The principle of the molecular beacon detection experiment is as follows: figure 1 As shown, the primer is complementary to the loop region of Molecular Beacon (MB, whose sequence is shown in SEQ ID NO.7), and after extension, the stem-loop of MB can be opened, resulting in luminescence. A stem-loop primer (ie Primer-MB shown in SEQ ID NO.8) was designed according to Molecular Beacon.

[0093] (1) Use 10×Reaction Buffer in EasyTaq DNA polymerase (TransGen, AP111) as the reaction solution, and prepare the reaction system in the PCR tube according to the following system:

[0094]

[0095] (2) Using a BioRad CFX96 fluorescent quantitative PCR instrument, incubate at...

Embodiment 3

[0102] Embodiment 3 Gene amplification and subtype identification of hybridoma cells

[0103] Design primers through the heavy chain and light chain of the antibody secreted by the hybridoma cell and the signal peptide sequence and constant region part of the primer, extract the total RNA of the hybridoma cell and reverse transcribe it into cDNA, use the cDNA as a template, use VH-VDJ-UmIgVH+( SEQ ID NO.9) and VH-VDJ-UmIgJH-(SEQ ID NO.10) amplified to obtain antibody heavy chain variable region (V H ) DNA sequence, using VL-VDJ-UmIgVK+ (SEQ ID NO.11) and VL-VDJ-UmIgJK- (SEQ ID NO.12) to amplify the antibody light chain variable region (V L ) DNA sequence.

[0104] Use 2×TransStart FastPfu Fly PCR SuperMix for amplification, and prepare the reaction system in the PCR tube according to the following system.

[0105]

[0106] Use a pipette or vortex to mix thoroughly, place the PCR tube in the PCR machine and perform the following procedures:

[0107]

[0108] The DNA se...

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Abstract

The invention discloses a Taq DNA polymerase monoclonal antibody combination as well as a reaction system and application thereof. The invention firstly discloses the Taq DNA polymerase monoclonal antibody combination. The combination comprises a monoclonal antibody 4A8 and a monoclonal antibody 9B7. The invention further discloses application of the antibody combination in reducing non-specific amplification of Taq DNA polymerase and / or ensuring thermal stability of Taq DNA polymerase, and a reaction system and a kit containing the antibody combination. The antibody combination and the optimized reaction solution can fully seal the active region of Taq DNA polymerase under the condition of low temperature, effectively reduce non-specific amplification and ensure the thermal stability of the enzyme, and can still effectively maintain the durability of the enzyme activity under the condition of higher amplification cycle number.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, it relates to a Taq DNA polymerase monoclonal antibody combination and its reaction system and application. Background technique [0002] Polymerase chain reaction (polymerase chain reaction, PCR) technology is widely used in molecular biology experiments, a DNA molecule can be amplified 10 times in a few hours in vitro 9 times. PCR technology is similar to the natural replication process of double-stranded DNA. Under the catalysis of DNA polymerase, the mother-strand DNA is used as the template, and the specific primer is used as the starting point of extension. Through steps such as denaturation, annealing, and extension, the DNA of the parent-strand template is replicated in vitro. Complementary daughter strand DNA process. DNA polymerase is the key to DNA replication. [0003] Taq DNA polymerase is a thermostable DNA polymerase isolated from Thermus aquatcus YT-1. ...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N9/96C12N9/12C12Q1/686
CPCC07K16/40C07K2317/56C07K2317/565C12N9/1252C12N9/96C12Q1/686C12Y207/07007C12Q2527/127C12Q2527/137
Inventor 曹洋李行宋新文张帅耿亮马静辛文
Owner BEIJING TRANSGEN BIOTECH CO LTD
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