Anti-serum interference Taq DNA polymerase and preparation and application thereof

A polymerase and antiserum technology, applied in the field of genetic engineering, can solve the problems of low anti-interference ability and insufficient polymerase activity, and achieve the effects of strong anti-interference ability, good thermal stability, and high enzyme expression and purification efficiency.

Active Publication Date: 2018-05-25
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the increasing popularization and application of PCR technology, researchers have continuously improved the performance requirements of Taq DNA polymerase, so researchers are constantly

Method used

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  • Anti-serum interference Taq DNA polymerase and preparation and application thereof
  • Anti-serum interference Taq DNA polymerase and preparation and application thereof
  • Anti-serum interference Taq DNA polymerase and preparation and application thereof

Examples

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Example Embodiment

[0040] Example 1: Preparation of Taq DNA polymerase gene expression vector against serum interference

[0041] Generally, the gene expression sequence can be selected according to the mutant Taq DNA polymerase that needs to be expressed, the corresponding gene sequence can be selected from the GeneBank, and mutation primers can be designed as needed to mutate one or a certain base sequence.

[0042] In this example, the gene expression sequence was prepared using the following operations:

[0043] 1. Obtain the nucleotide sequence shown in SEQ ID NO:1 and clone the sequence

[0044] Sampling location: Rehai, Tengchong City, Yunnan Province (1390m above sea level)

[0045] Mixed soil samples: Huaitaijing (88℃), Pearl Spring (98℃), Guming Spring (85℃), Toadzui No. 4 Hillside Soil (79.9℃), mix well and take 10g for use.

[0046] Water samples: Toad mouth No. 1 (65.2°C), Toad mouth No. 3 (88°C), Toad mouth No. 4 hillside running water (79.9°C), pregnant well left (91.7°C), pregnant well righ...

Example Embodiment

[0054] Example 2: Expression and purification of Taq DNA polymerase interfered by antiserum

[0055] Refer to the method recommended by the kit manufacturer for the transformation of the gene expression vector. The host cell is a competent cell, such as E. coli competent cell. The constructed gene expression vector is added to the competent cell and heat shocked to make the competent cell The cell membrane structure is disturbed, and there are gaps in the cell membrane for the gene expression vector to enter the cell, and then the cell is cultured at a constant temperature to revive the host cell. The specific method for the expression and purification of Taq DNA polymerase interfered by antiserum in this embodiment is as follows:

[0056] The Taq DNA polymerase gene expression vector (pET28a-Taq DNA) obtained by metagenomic analysis was transformed into E. coli competent cells BL21. Resistance Screening Pick a single clone and culture it in 5mL LB liquid medium at 37°C at 180rpm ...

Example Embodiment

[0057] Example 3: SDS-PAGE protein electrophoresis to verify the protein size and purity of Taq DNA polymerase against serum interference

[0058] The Taq DNA polymerase obtained above is subjected to SDS-PAGE protein electrophoresis detection to identify the size and purity of the mutant Taq DNA polymerase protein.

[0059] The concentration of SDS-PAGE concentrated gel is 5%, and the concentration of separation gel is 8%. Take 40 μL of the dialyzed protein sample and mix with 10 μL of 5× protein loading buffer and boil for 10 minutes for sample preparation. After the sample is prepared, take 10μL, load it together with the protein Marker (ProteinRuler II(12-120kDa), Thermo Scientific Fermentas), and electrophoresis at a constant voltage of 50V for 30min. When the protein sample is electrophoresed to the interval between the concentrated gel and the separating gel, change the voltage to 150V constant electrophoresis to the bottom of the separation gel. After the electrophoresis ...

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Abstract

The invention relates to an anti-serum interference Taq DNA polymerase analyzed and obtained from a hot-spring meta genome, and relates to the anti-serum interference Taq DNA polymerase and a preparation method and application thereof. The protein amino acid sequence of the anti-serum interference Taq DNA polymerase is shown as SEQ ID No:1; the genes of the anti-serum interference Taq DNA polymerase are coded, and the nucleotide sequence is shown as SEQ ID No:2. Compared with the amino acid sequence of a wild Taq DNA polymerase, the protein amino acid sequence of the polymerase has 13 amino acid mutations. The sequence is cloned and subjected to polymerase expression and purification, and the polymerase has higher polymerase activity than a commercial Taq DNA polymerase and the wild Taq DNA polymerase; the polymerase is used for simulated sample detection in serum, and the result of a fluorogenic quantitative PCR shows that the polymerase has better tolerance than the wild Taq DNA polymerase and the commercial polymerase. Therefore, the polymerase can be used for conducting direct PCR detection on a nucleic acid extraction-free serum sample.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an anti-serum interference Taq DNA polymerase obtained from hot spring metagenome analysis, and to a preparation method and application of the anti-serum interference Taq DNA polymerase. Background technique [0002] Taq DNA polymerase is a heat-resistant DNA polymerase isolated by scientist A.Chien from the extreme thermophile Thermus aquaticus in 1976 (Chien A, Edgar DB, Trela ​​JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.[J ]. Bacteriol, 1976, 127(3): 1550-1557.). In 1983, Kary Mullis invented PCR (polymerase chain reaction). In 1988, Randall K. Saiki and others applied Taq DNA polymerase to PCR technology for the first time, making the PCR process realize automatic continuous cycle. Taq DNA polymerase is a heat-resistant DNA polymerase belonging to the DNA polymerase I family. The enzyme gene is 2496 bases in length, encodi...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70
CPCC12N9/1252C12Y207/07007
Inventor 危宏平余军平熊进张晓旭
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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