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Recombinant Taq DNA polymerase and preparation method thereof

A technology of polymerase and lysozyme, applied in the field of genetic engineering

Active Publication Date: 2014-02-26
哲尔基因科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]Although Taq DNA polymerase has been commercially produced for many years, in order to increase the expression of the protein in Escherichia coli, attempts to simplify the purification process have not stopped

Method used

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  • Recombinant Taq DNA polymerase and preparation method thereof
  • Recombinant Taq DNA polymerase and preparation method thereof
  • Recombinant Taq DNA polymerase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Cloning and analysis of the full-length coding region of the gene

[0022] A commercially available bacterial genome DNA extraction kit was used to extract Thermus aquaticus ( Thermus aquaticus ) total DNA, using Touchdown PCR technology and ordinary PCR technology to amplify the full-length sequence of Taq DNA polymerase gene, the amplification primers include 2 sets (5'-tatatgagggggatgctgccc-3' and 5'-tcactccttggcggagagc-3'). After the PCR products were detected by 1% agarose gel electrophoresis, the gel blocks containing the target bands were cut under ultraviolet light, and the target fragments were recovered using an agarose gel recovery kit, and stored at -20°C. The recovered target fragment was connected to the cloning vector pMD19-T (TaKaRa) in a metal bath at 16°C overnight, and transformed into Escherichia coli competent cells E. coli In Top10 (Beijing Tiangen Biochemical Technology Co., Ltd.), spread on LB solid medium containing 100mg / mL Amp, c...

Embodiment 2

[0023] Embodiment 2: Cultivation and induction of recombinant Taq DNA polymerase strain

[0024] Prepare LB liquid shake flasks (500 ml of LB liquid medium, divided into two 500 ml Erlenmeyer flasks, that is, 250 ml in each bottle, sterilized for use). Take out the Taq DNA polymerase strain, inoculate it in 4 ml of LB + Amp (100 μg / ml) liquid medium, and incubate at 37°C for 16-20 hours. Fill each Erlenmeyer flask with 250 ml of LB liquid medium, add Amp (the final concentration of antibiotics is 100 μg / ml), and inoculate with 1.25 ml of cultured bacteria; incubate at 37°C for 4.5 hours.

[0025] Add IPTG at a final concentration of 0.5 mM for induction, and continue culturing at 37° C. for 16-20 hours.

Embodiment 3

[0026] Example 3: Recombinant Taq DNA polymerase purification equipment preparation

[0027] The chromatographic column uses Bio-Rex 70 ion exchange column resin (the size of the dry powder is mesh size 100-200. The binding capacity after swelling is 5-10 mg protein / ml). Weigh 7g of dry resin of Bio-Rex 70, put it in a small beaker, wash it with dd H2O, remove the small floating particles, swell at room temperature for 2 hours, change dd H several times during the period 2O; pre-equilibrated with solution C containing 50 mM KCl, ready for use; equilibrated resin with solution C containing 50 mM KCl. After loading the resin, equilibrate the column with 6-10 column bed volumes of Solution C containing 50 mM KCl. Check that the pH of Solution C does not change as it enters and exits the column.

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Abstract

The invention relates to the technical field of genetic engineering, and particularly relates to a method for preparing a gene recombinant expression enzyme of Thermusaquaticus TaqDNA polymerase. The nucleotide sequence of a gene and the amino acid sequence of an encoded protein are respectively shown as SEQIDNO:1 and SEQIDNO:2. According to the invention, the method comprises the following steps: cloning a gene sequence by using a gene cloning technique, and building a prokaryotic expression vector; by using a recombinant strain of a built TaqDNA polymerase gene, carrying out suspension by using an LB culture solution; after carrying out culturing for 4.5 hours at a temperature of 37 DEG C, adding IPTG (isopropyl-beta-d-thiogalactoside) with a final concentration of 0.5 mM into the obtained substance to induce for 16-20 hours; after thalli are collected in a centrifugal mode, dissociating the thalli by using 2-4 mg / ml lysozyme, so that protein fluid is obtained; and filtering the protein fluid by using a chromatographic column and a dialysis bag so as to obtain high-purity active protein TaqDNA polymerase. Compared with the prior art, stability of a production process is good, activity of obtained TaqDNA polymerase is high, the yield and purity of products are effectively ensured, and amplification effect when being applied to a gene PCR (polymerase chain reaction) is good.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing recombinant Taq DNA polymerase. Background technique [0002] In 1985, the American Cetus Company invented the epoch-making polymerase chain reaction (polymerase chain reaction, PCR), which is a method for rapid amplification of specific DNA fragments in vitro, that is, double-stranded DNA is denatured at high temperature Melt into a single strand, and with the participation of DNA polymerase, dNTP, and specific primers, copy into the same two molecular copies according to the principle of complementary base pairing. [0003] The original bacteria YT-1 (Thermus aquatjcus) has a natural DNA polymerase, but its Taq DNA polymerase content is low, and the purification of the enzyme from its culture requires selective precipitation and ion exchange chromatography separation, which is difficult to meet the demand for Taq DNA polymerase . Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12R1/01
CPCC12N9/1252C12N15/70C12Y207/07007
Inventor 刘涛马秀芬
Owner 哲尔基因科技(上海)有限公司
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