Novel promoter and application thereof

A promoter, a new type of technology, applied in the direction of using vectors to introduce foreign genetic material, DNA/RNA fragments, microorganisms, etc., can solve the problem that the subtilis expression system is not very mature

Active Publication Date: 2015-07-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The gene expression system is a complicated process, and the subtilis expression system is not very mature at present

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: pMA0911P HpaII -Construction of pro-NK vector

[0045] According to the nattokinase gene sequence (Genbank accession number: S51909.1) provided by Genbank, primers pro-NK-F / pro-NK-R were designed, and the genomic DNA of Bacillus natto (B.natto) was used as a template for amplification pro-nk gene, the gel electrophoresis band is about 1100bp, which is consistent with the size of the pro-nattokinase gene; (2) the PCR product pro-nk is purified, and after double digestion with EcoRI and BamHI, it is connected to On the Escherichia coli-Bacillus subtilis shuttle plasmid pMA0911-wapA double-digested with endonuclease, the recombinant expression plasmid pMA0911P was obtained by ligation HpaII -pro-NK, transform the plasmid into Escherichia coli JM109, and send it for sequencing after digestion and verification. The sequencing results show that the pronattokinase gene was successfully inserted into the vector pMA0911-wapA; (3) extract the plasmid and transform it ...

Embodiment 2

[0049] Embodiment 2: the design of new promoter gene sequence

[0050] promoter P d(-35～-10)HpaII The design of the gene sequence:

[0051] (1) According to P HpaII The core element of promoter gene sequence -35 region (TTTATG) and -10 region (TATACT) design primer P d(-35～-10)HpaII -F / P d(-35～-10)HpaII -R, to pMA0911P HpaII -pro-NK plasmid was used as a template for full-plasmid PCR, and the obtained PCR product was treated with DpnⅠ and then transformed into Escherichia coli JM109 for plasmid amplification. After coating a plate containing resistance, it was cultured at 37°C, and a single colony was picked and transferred to a plate containing Resistant liquid 2×YT medium culture;

[0052] (2) The extracted plasmid was verified by PCR with primers pMA-F / pMA-R ( figure 1 ), figure 1 The size of the middle band is 364bp, which is consistent with the size of the predicted result. DNA sequencing was carried out at the same time. The DNA sequencing results showed that the ...

Embodiment 3

[0054] Embodiment 3: fermentative synthesis of recombinant nattokinase

[0055] Construction of recombinant bacteria: transfer the recombinant plasmid with the correct sequence obtained in Example 2 into Bacillus subtilis WB800, and after standing overnight at 37°C, pick a single colony and place it in 10mL 2×YT seed medium at 37°C culture; according to the final OD 600 0.02 was transferred to a 250mL Erlenmeyer flask containing 30mL TB medium, 200r / min, temperature 37°C, and cultured for 36 hours. The fermentation supernatant was taken for SDS-PAGE protein electrophoresis ( figure 2 ).

[0056] Such as image 3 As shown, the electrophoresis band of about 28kDa was obtained, indicating that the recombinant Bacillus subtilis successfully secreted and expressed nattokinase. The fibrinolytic activity of recombinant nattokinase was roughly measured on fibrin plate ( image 3 ), the original strain could not hydrolyze fibrin, while the fermentation supernatant of the recombin...

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Abstract

The invention discloses a novel promoter and application thereof, and belongs to the field of microbial genetic engineering. According to a method disclosed by the invention, the related promoter Pd(-35 to -10) HpaII gene is formed by series connection modification on the core area based on the promoter PHpaII; under control and transcription of the novel promoter Pd(-35 to -10) HpaII, the fibrinolytic activity of the recombinant nattokinase is up to 200.8 FU/ml. The method is efficient, safe and capable of effectively improving the expression quantity of nattokinase, and provides a theoretical basis for further study on influence of the promoter on heterologous gene expression.

Description

Technical field: [0001] The invention relates to a novel promoter and its application, belonging to the field of microbial genetic engineering. Background technique: [0002] Nattokinase (Nattokinase, NK) is an alkaline serine protease produced by Bacillus natto or Bacillus subtilis natto. Because of its special thrombolytic activity, it can prevent and treat vascular embolism diseases. Compared with drugs, it has the advantages of good safety performance, non-immunogenicity, easy to be digested and absorbed by the human body, can be injected intravenously or orally, has a long half-life, and is cheap to produce. drug. However, due to the low enzyme production of nattokinase wild bacteria, its development is severely limited, so increasing the enzyme production of nattokinase has good research value. [0003] The Bacillus subtilis expression system is considered to be a GRAS-level organism. It has no obvious codon preference, can secrete functional proteins into the medium...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/75C12N1/21C12N9/54C12N9/48C12R1/125
Inventor 周哲敏刘中美崔文璟周丽葛春蕾
Owner JIANGNAN UNIV
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