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Sso7d-Sau recombinant DNA polymerase

A polymerase and N-terminal technology, applied in the fields of molecular biology and protein engineering, can solve the problem of low continuous synthesis ability of DNA polymerase, achieve high tolerance, wide application prospects, and improve the effect of DNA amplification

Inactive Publication Date: 2015-04-01
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to provide a kind of Sso7d-Sau recombinant DNA polymerase, is to be derived from the DNA binding protein Sso7d of thermophilic bacterium Sulfolobus solfataricus covalently linked at the N-terminus of Staphylococcus aureus DNA polymerase I (Sau), strives to overcome The problem of low processivity of most DNA polymerases in the DNA polymerase I family

Method used

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  • Sso7d-Sau recombinant DNA polymerase
  • Sso7d-Sau recombinant DNA polymerase
  • Sso7d-Sau recombinant DNA polymerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example is used to illustrate the acquisition of Sso7d-Sau fragments.

[0036] 1. Synthesize the Sso7d-linker sequence as shown in SEQ ID No.3, wherein the Sso7d sequence refers to the sequence information published in GenBank (NCBI reference sequence number is NC_002754.1), and the linker sequence is GGAGGCGGAGGCTCA GGAGGCGGAGGCTCA GGAGGCGGAGGCTCA.

[0037] 2. Design and synthesize primers for Sso7d-linker sequence and Sau gene sequence published by GenBank (NCBI reference sequence number is YP_006237943.1):

[0038] Sso_F: 5'-CATG CCATGG CAACAGTAAAGTTCAA-3'(NcoI) (SEQ ID No.4)

[0039] Linker_R: 5'-GTCGACGCTTGCTGATGAGCCTCCG-3' (SEQ ID No.5)

[0040] PS_F: 5'-CGGAGGCTCATCAGCAAGCGTTG-3' (SEQ ID No.6)

[0041] PS_R: 5'-TTTTCCTTTT GCGGCCGC TTTTGCATCATACCAAGTTGC-3'(NotI) (SEQ ID No.7)

[0042] 3. Use specific primers Sso_F / Linker_R to use the artificially synthesized plasmid pUC57-Sso7d-linker containing the Sso7d-linker gene as a template for PCR amplification; ...

Embodiment 2

[0057] This embodiment is used to illustrate the construction of recombinant plasmid pTrc99A-Sso7d-Sau ( figure 2 ).

[0058] 1. Sso7d-Sau fragment and pTrc99A vector were digested at 37°C for 2 hours, and the digested products were recovered by gel. The enzyme digestion system is as follows:

[0059]

[0060] 2. Ligate the digested Sso7d-Sau and pTrc99A fragments with T4 ligase, and ligate overnight at 16°C. The connection system is as follows:

[0061]

[0062] 3. Transformation: Take out the Escherichia coli DH5α competent cells from the -70°C refrigerator and bury them in ice cubes, take out the ligation product from the 16°C water bath, remove the parafilm, and put them on ice. After the competent cells melt, put Add all the ligation products to 100 μl DH5α competent cells and mix well. On ice for 30 minutes. Heat shock at 42°C for 90s, and act on ice for 2min. Then add 800 μl LB, shake at 180 r / min for 1 hour at 37°C. The culture was centrifuged at 5000r / mi...

Embodiment 4

[0082] This example illustrates the induced expression and purification of recombinant proteins.

[0083] 1. Transform the positive recombinant expression plasmid pTrc99A-Sso7d-Sau verified by sequencing into Escherichia coli expression strain BL21(DE3), pick a single colony and culture in LB liquid medium containing kanamycin overnight at 37°C .

[0084] 2. Inoculate the overnight bacterial solution into 500ml LB liquid medium containing kanamycin at a ratio of 1 / 100, and culture it on a shaker at 37°C until OD 600 When the value reached 0.6, IPTG was added to a final concentration of 0.5 mmol / L for induction. At the same time, the expression vector control group was set, and the bacterial liquid was taken 4 hours after induction of expression, centrifuged at 12 000 r / min for 10 minutes, and the supernatant was removed.

[0085] 3. Ni-NTA sample buffer (20mM Na 3 PO 4 , 0.5M NaCl, 10mM imidazole, pH 7.4) to resuspend the bacterial pellet, after ultrasonic disruption, cent...

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Abstract

The invention relates to an Sso7d-Sau recombinant DNA polymerase. The Sso7d-Sau recombinant DNA polymerase is the hybrid DNA polymerase obtained by covalently linking Sso7d protein to the N end of a staphylococcus aureus DNA polymerase I (Sau) protein sequence by a flexible linkage sequence, wherein the amino acid sequence of the Sso7d-Sau recombinant DNA polymerase is shown in SEQ ID No. 2, or an amino acid sequence is formed by replacing, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID No. 2 and has the same function as the amino acid sequence shown in SEQ ID No. 2. The invention further provides a gene for coding the protein as well as a preparation method and an application of the protein. The transformed DNA polymerase disclosed by the invention keeps the catalytic activity invariable, however, the processivity is remarkably improved, the tolerance to salt is improved, and the DNA amplification effect of a recombinase-mediated nucleic acid isothermal amplification technology can be remarkably improved during farmland and field detection processes. The DNA polymerase has a wide application prospect.

Description

technical field [0001] The invention belongs to the fields of molecular biology and protein engineering, and relates to a Sso7d-Sau recombinant DNA polymerase. Background technique [0002] Nucleic acid amplification is a very important technology in biological research, medical testing, forensic identification, agriculture and other related fields. Currently, the most widely used nucleic acid amplification technique is the polymerase chain reaction (Polymerase Chain Reaction, PCR). PCR technology uses cyclical changes in reaction temperature to achieve denaturation, renaturation and extension of the target sequence, and can achieve exponential amplification of the target sequence within 2-3 hours. However, because PCR technology relies on PCR instruments with large volume, high power consumption and high price, its wide application in on-site and field testing is limited. [0003] In recent years, there have been many research advances in the field of molecular biology of...

Claims

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Application Information

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IPC IPC(8): C12N9/12C07K14/195C12N15/62C12N15/70C12N15/10
CPCC12N9/1252C07K14/195C07K2319/80C12Y207/07007
Inventor 程奇翟冰周国辉李贤祯刘国宪
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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