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Strong promoter and application of strong promoter to improving expression of nattokinase

A strong promoter and promoter technology, applied in the direction of enzymes, peptidases, hydrolytic enzymes, etc., can solve the problem that the production of nattokinase is insufficient to meet production needs, and achieve good results

Active Publication Date: 2016-04-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current output of nattokinase is still not enough to meet the production demand

Method used

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  • Strong promoter and application of strong promoter to improving expression of nattokinase
  • Strong promoter and application of strong promoter to improving expression of nattokinase
  • Strong promoter and application of strong promoter to improving expression of nattokinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: pMA0911P HpaII -Construction of pro-NK vector

[0044] 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 nattokinase gene was successfully inserted into the vector pMA0911-wapA; (3) extract the plasmid and transform it into...

Embodiment 2

[0047] Embodiment 2: the design of strong promoter gene sequence

[0048] promoter P HpaII- P HpaII The design of the gene sequence:

[0049] (1) According to P HpaII Promoter gene sequence design primer P HpaII- P HpaII -F / P HpaII- P HpaII -R, to pMA0911P HpaII -Pro-NK plasmid was used as a template for PCR, and the resulting PCR product was used as a primer, pMA0911P HpaII -Pro-NK plasmid was used as a template for large primer 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;

[0050] (2) DNA sequencing, the sequencing results show that the target gene fragment is successfully inserted into the original vector P HpaII Behind the gene sequence, a new promoter P is formed HpaII -P HpaII (The nucleotide sequenc...

Embodiment 3

[0052] Embodiment 3: fermentative synthesis of recombinant nattokinase

[0053] 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 Cultivate; transfer to a 250mL Erlenmeyer flask containing 30mL TB medium according to the inoculation amount of 1-5%, 200r / min, temperature 37°C, and cultivate for 48 hours. The fermentation supernatant was taken for SDS-PAGE protein electrophoresis ( image 3 ).

[0054] 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 each recombinant nattokinase was detected by ultraviolet spectrophotometer ( Figure 4 ), the specific results are shown in Table 2. The results showed that the expressi...

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Abstract

The invention discloses a strong promoter and an application of the strong promoter to improving expression of nattokinase, and belongs to the field of microorganism genetic engineering. The genes of the strong promoter in the series of technical schemes are formed in the mode that a promoter PP43 and / or PHpa II are / is subjected to n (n is larger than or equal to 1) times of series connection. Under transcription control of a strong promoter PHpa II-PHpa II-PP43, the fibrinolysis activity of recombined nattokinase is as high as 264.2 FU / m1. A method is efficient and safe, the expression quantity of nattokinase can be effectively increased, and a theoretical foundation is provided for further researching the influence of the promoter to the expression of heterologous genes.

Description

technical field [0001] The invention relates to a strong promoter and its application in improving the expression of nattokinase, belonging to the field of microbial genetic engineering. Background technique [0002] Cardiovascular and cerebrovascular diseases are a serious threat to human health, and the current clinically injected antithrombotic drugs generally have disadvantages such as poor stability, short efficacy, large side effects, and high cost. Since 1987, Japanese scholar Sumi et al. isolated and purified nattokinase with high thrombolytic effect from natto for the first time, and it has become one of the new research directions of thrombolytic drugs in recent years. Nattokinase (Nattokinase, NK) is an alkaline serine protease produced by Bacillus natto or Bacillus subtilis. Because of its special thrombolytic activity, it can prevent and treat thrombosis. Compared with other thrombolytic drugs, it has the advantages of good safety performance, no immunogenicity...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/75C12N1/21C12N9/54C12N9/48
CPCC12N9/485C12N9/54C12Y304/11011
Inventor 周哲敏刘中美崔文璟周丽葛春蕾
Owner JIANGNAN UNIV
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