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Fusaruside producing engineering bacterium, and construction method and applications thereof

A construction method and technology of engineering bacteria, applied in the field of microbiology and medicine, can solve the problems that restrict the research and application of cerebroside immunosuppressants, and achieve the effect of shortening the production cycle and simple method

Active Publication Date: 2018-11-13
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These factors seriously restrict the research and application of cerebroside immunosuppressants

Method used

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  • Fusaruside producing engineering bacterium, and construction method and applications thereof
  • Fusaruside producing engineering bacterium, and construction method and applications thereof
  • Fusaruside producing engineering bacterium, and construction method and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment one: the acquisition and transformation of target gene

[0038] The 3-position desaturase (Δ3(E)-Sphingolipid Desaturase, Δ3(E)-SD) and 10-position desaturase (Δ10(E)-SD) genes required in the present invention are all from Fusarium graminearum Fusarium graminearum PH-1. The strain was purchased from the American Culture Collection, its total RNA was extracted, cDNA was obtained by reverse transcription, and the Δ3(E)-SD gene (1722bp) and Δ10(E)-SD gene (1320bp) were amplified by PCR. The 2A peptide used for co-expression is the most commonly used F2A, derived from foot-and-mouth disease virus (FMDV), the gene is obtained by chemical synthesis, the length is 66bp, and the nucleotide sequence is as shown in SEQ ID NO. 13.

[0039] In the present invention, the pPIC3.5K vector is selected for the double-gene co-expression experiment. Since the number of restriction sites is small, and the target gene and the F2A gene contain these restriction sites, the targe...

Embodiment 2

[0042] Embodiment two: the construction of co-expression vector

[0043] The present invention uses OL-PCR method to insert 2A peptide gene between two desaturase genes (Δ3(E)-SD and Δ10(E)-SD) to form Δ3(E)-SD-2A-Δ10( E) The combined gene fragment of-SD, using BamH I and EcoR I to double digest the combined fragment and the yeast expression vector pPIC3.5K, then connect and transform into Escherichia coli. PCR verification was performed on the grown E. coli colonies. After extracting the plasmid from the positive bacteria, carry out enzyme digestion verification, the schematic diagram of the constructed co-expression vector and the enzyme digestion verification results are as follows: figure 1 As shown, a fragment with a length of about 3000bp can be obtained after enzyme digestion, which is consistent with the length of the combined gene fragment (3108bp), indicating that the dual-enzyme co-expression vector was successfully obtained.

Embodiment 3

[0044] Embodiment three: transformation and transformant screening of recombinant vector

[0045] Conversion:

[0046] In the present invention, the constructed co-expression vector (about 10 μg) is digested with Bgl I to linearize the target gene. Linearized vectors are dephosphorylated with alkaline phosphatase to prevent self-ligation. At the same time, prepare Pichia competent cells according to the following method:

[0047] 1. Pichia pastoris was inoculated in 500mL YPD medium, cultured overnight at 30°C, so that the bacterial concentration reached 5-7x 10 7 ;

[0048] 2. Collect the cells by centrifugation at 3000g at 4°C for 5 minutes;

[0049] 3. Add 50mL YPD / HEPES, 1.25mL 1M DTT, mix gently, incubate at 30°C for 15min, add 200mL pre-cooled 1M sorbitol, centrifuge at 3000g at 4°C for 5min to collect cells;

[0050] 4. Wash with 250mL pre-cooled 1M sorbitol for 2 times, and 10mL pre-cooled 1M sorbitol for 1 time;

[0051] 5. Finally, resuspend in 0.5mL of pre-coo...

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Abstract

The invention belongs to the technical field of microorganism and medicine technology, and more specifically relates to a fusaruside producing engineering bacterium, and a construction method and applications thereof. According to the construction method, expression of the 3th site desaturase and the 10th side desaturase in fusarium graminearum is carried out, so that a pichia pastoris bacterial strain capable of producing fusaruside is constructed. Compared with the fusarium used for original production of fusaruside, the pichia pastoris bacterial strain possesses following advantages: the fermentation time is shorter, only 5 to 7 days are needed, while 10 to 14 days are needed by the fusarium. The fusaruside producing engineering bacterium is capable of solving a problem that the contentof fusaruside in fusarium is low, the yield is increased by 11.6 times of that of the fusarium, and foundation is provided for further development and applications of fusaruside.

Description

technical field [0001] The invention relates to the technical fields of microorganisms and medicine, in particular to a fusaruside-producing engineering bacterium and its construction method and application. Background technique [0002] Immunosuppressants are the main drugs in the treatment of transplant rejection and autoimmune diseases. However, the immunosuppressants commonly used in clinical practice currently have many adverse reactions, and long-term use will lead to serious side effects, which are mostly related to the low selectivity of immunosuppressive drugs for targets. Therefore, it is urgent to find new therapeutic targets for immune system diseases and to develop new small molecule immunosuppressants with high efficiency, low toxicity and high selectivity for these targets. [0003] Recent studies have found that the novel cerebroside small molecule compound fusaruside has good immunosuppressive activity, which can specifically inhibit the activation of STAT1...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P19/44C12R1/84
CPCC12N9/0004C12N15/815C12P19/44
Inventor 田园李艳玲
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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