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Modified strain, application of modified strain in preparation of intestinal motility promoting preparation and product

An intestinal motility and strain technology, applied in the biological field, can solve the problems of obvious side effects, unstable effect, unclear mechanism, etc., and achieve the effects of reducing side effects, controllable expression, and precise control of drug delivery.

Pending Publication Date: 2021-12-31
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Intestinal motility-promoting preparations of small-molecule drugs have obvious side effects and cannot be administered repeatedly for a long time. They are not suitable for patients with long-term and repeated constipation attacks
However, probiotic intestinal motility preparations, due to the unclear mechanism, have unstable effects in relieving constipation and often vary from person to person

Method used

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  • Modified strain, application of modified strain in preparation of intestinal motility promoting preparation and product
  • Modified strain, application of modified strain in preparation of intestinal motility promoting preparation and product
  • Modified strain, application of modified strain in preparation of intestinal motility promoting preparation and product

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 expression vector construction

[0037] The pBAD-hTPH1 expression vector is constructed according to the following method:

[0038] S1 Obtain the target gene fragment: obtain the target gene fragment by artificial synthesis according to the preferred expression sequence of tryptophan hydroxylase TPH1.

[0039] S2 Obtain the target vector fragment: Use restriction enzymes ApaLI and EcoRI to double digest the plasmid pBAD24. The enzyme digestion reaction system is: 10μLCutsmart, 2μLApaLI, 2μLECoRI, 2μg plasmamid, ddH 2 O less than 50 μL; 1% agarose gel electrophoresis detection after the reaction, such as figure 2 As shown, use the AxyprepDNA Gel Extraction kit 50-prep gel extraction kit to recover large fragments of the target vector fragment.

[0040] S3 to obtain the recombinant vector: using the GoldenGate cloning method, the reaction system: ApaLI-HF0.75ul, EcoRI-HF 0.75ul, 10×CutSmart TMBuffer 1ul, ATP (10mM) 1ul, T7 DNA ligase0.25ul, target gene fr...

Embodiment 2

[0042] Embodiment 2 obtains the transformation bacterial strain EcN pTPH bacterial strain

[0043] S1 recombinant vector construction: same as Example 1.

[0044] S2 Extract the recombinant vector: inoculate the successfully verified VB UltraStable strain containing the recombinant plasmid in 4 mL of LB culture medium containing ampicillin antibiotic (100 ng / mL), and culture at 37° C. and 180 rpm for 24 hours. Plasmids were extracted using the Novizan plasmid mini-prep kit.

[0045] Obtain the modified strain in S3: Electrotransform the recombinant plasmid obtained in S2 into EcN competent cells. The transformation method is carried out according to electrotransformation. After 1 h, the bacterial cells were obtained by centrifuging and concentrating. Spread the bacteria on the ampicillin antibiotic (100ng / mL) plate, culture for 24h, and obtain a positive strain, which is the modified strain.

[0046] Following embodiment 3-5, schematic flow sheet is as figure 2 As shown, ...

Embodiment 3

[0049] Embodiment 3 Feces water content test

[0050] After the end of gavage, put the mice individually into a cage lined with absorbent paper, collect the feces of 3 mice, weigh them as the wet weight, and after freeze-drying, it is the dry weight, according to the following formula:

[0051] Water content of feces = water content of feces (%) = (wet weight of feces - dry weight of feces) / wet weight of feces × 100, calculate the water content of feces, and take the average value.

[0052] The result is as image 3 As shown in Table 1:

[0053] Table 1 Fecal water content test results

[0054] normal group(%) Model group (%) EcN wild type (%) EcNpTPH (%) 66.86909 50.26911 57.54977 59.33918 68.03653 53.16117 55.73154 61.59484 66.5937 54.73606 54 61.87923

[0055] Shows: normal group, model group, EcN wild-type bacteria group, EcN pTPH bacteria group feces moisture content are: 67.15%, 52.72%, 55.76%, 60.94%

[0056] It can be seen...

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Abstract

The invention discloses a modified strain, application of the modified strain in preparation of an intestinal motility promoting preparation and a product. The modified bacterium has an expression vector, and an expression region of the expression vector comprises a tryptophan hydroxylase TPH1 expression sequence. The modified strain provided by the invention is colonized in an intestinal environment through probiotics, continuously expresses tryptophan hydroxylase, and further continuously catalyzes to generate 5-hydroxytryptamine, so that intestinal motility is stably and continuously promoted, a stable and reliable constipation relieving effect is provided, and the modified strain can be used for preparing intestinal motility promoting preparations and constipation relieving preparations.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a modified bacterial strain, its application in preparing enterokinetic preparations and products. Background technique [0002] At present, it has been found that some substances can act on the enteric nerves, cause smooth muscle contraction, increase intestinal motility, shorten intestinal transit time, reduce water recovery in feces, and have the effect of relieving constipation, that is, intestinal motility preparations. [0003] The existing intestinal motility-promoting preparations mainly include small-molecule drugs, such as mosapride: it acts on the serotonin receptors of the enteric nerves to promote gastrointestinal motility; The action of enzymes promotes the motility of the gastrointestinal tract; Prokapril: serotonin receptor agonist, stimulates serotonin receptors, causes high-amplitude propulsive contraction of the intestinal tract, and accelerates col...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/70C12N15/65A61K48/00A61K38/44A61P1/10A61P1/14C12R1/19
CPCC12N9/0071C12N15/70C12N15/65C12Y114/16004A61K48/0008A61K48/005A61K38/44A61P1/10A61P1/14C12N2800/22C12N2800/101
Inventor 刘智李蓓罗亚楠
Owner HUAZHONG UNIV OF SCI & TECH
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