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Staphylococcus sciuri CY1-78 and application thereof in preparation of fibrinolysin by fermentation

A technology of Staphylococcus squirrel and fibrinolytic enzyme, which is applied in the biological field, can solve the problems of short half-life, invalid oral administration and high price, and achieves the effects of easy cultivation, simple separation and purification process, and short fermentation period.

Active Publication Date: 2021-02-02
ZHEJIANG SHUREN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are some shortcomings in the clinical use of fibrinolytic drugs, such as easy to cause bleeding, short half-life, oral ineffectiveness, high price, etc.

Method used

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  • Staphylococcus sciuri CY1-78 and application thereof in preparation of fibrinolysin by fermentation
  • Staphylococcus sciuri CY1-78 and application thereof in preparation of fibrinolysin by fermentation
  • Staphylococcus sciuri CY1-78 and application thereof in preparation of fibrinolysin by fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Isolation and screening of strains producing fibrinolytic enzyme

[0035] The wild strain isolation and screening method of described Staphylococcus squirrel CY1-78 bacterial strain are as follows:

[0036] (1) Plate separation: cut a silkworm cocoon, take out the silkworm chrysalis, soak it in 75% ethanol aqueous solution at room temperature (about 25°C) for 1min, take it out, cut off the head, open the abdominal cavity and take out the midgut; In a sterile mortar, add 1 mL of sterile saline to grind until homogenized; dilute the homogenate with sterile physiological water to 1×10 4 – 1×10 6 times. Draw 0.1mL of 1×10 5 , 1×10 6 , 1×10 7The 3 times dilution solution was spread on the skim milk plate medium, and cultivated in a 37°C incubator until the number of colonies no longer increased. Pick the colony with a transparent circle around it, streak and inoculate it on a fresh LB plate medium, culture at 37°C until a single colony grows, and then pick a...

Embodiment 2

[0049] Example 2: Classification and identification of enzyme-producing strains

[0050] The culture characteristics of the CY1 strain are as follows: the strain was inoculated on an LB plate, and after culturing at 37°C for 24 hours, milky white colonies could be seen, the colonies were opaque, with neat edges and a smooth surface (see the attached photo for the colony photos). figure 1 ). The CY1 strain was inoculated on the skimmed milk plate medium, and after culturing at 37°C for 24 hours, a large transparent circle was formed around the colony (see the attached photo for the colony photo). figure 2 ).

[0051] The cell characteristics of the CY1 strain are as follows: Gram-positive, spherical, and 0.8-1.2 μm in diameter. Bacteria are often distributed singly or in irregular clusters, and the optical microscope photos after Gram staining are shown in the appendix image 3 .

[0052] The total DNA of the CY1 strain was extracted, and the 16S rDNA fragment of the strai...

Embodiment 3

[0054] Mutation breeding of embodiment 3 Staphylococcus squirrel

[0055] The CY1 strain of Staphylococcus squirrel was subjected to ultraviolet mutagenesis, and the CY1-78 strain of Staphylococcus squirrel was obtained by screening.

[0056] (1) Cell preparation: 20 mL of LB liquid medium was inserted into slanted cells of Staphylococcus squirrel CY1 strain, and cultured at 37° C. and 200 r / min shaking for 12 h. Take 1mL of the bacterial solution in a centrifuge tube, centrifuge at 5000r / min for 5min, discard the supernatant, add 1mL of sterile saline to resuspend the bacterial cells, and centrifuge again to collect the bacterial cells. Resuspend the bacteria in normal saline in a 100mL Erlenmeyer flask for later use. The composition and preparation method of the LB liquid medium are the same as in Example 1.

[0057] (2) Ultraviolet mutagenesis: Take 6 pairs of sterile petri dishes with a diameter of 6 cm, and add 1 mL of the above bacterial suspension respectively. Open ...

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Abstract

The invention discloses Staphylococcus sciuri CY1-78 and application thereof in preparation of fibrinolysin by fermentation. The Staphylococcus sciuri CY1-78 strain is inoculated into an enzyme production medium, fermentation is performed at 30-35 DEG C and 200-250 r / min for 24-32 hours to obtain a fermentation broth, the fermentation broth is centrifuged, thalli are removed to obtain a crude fibrinolysin solution, and salting-out, dialysis and freeze-drying are performed to obtain fibrinolysin freeze-dried powder. The Staphylococcus sciuri CY1-78 strain capable of producing the fibrinolysin,provided by the invention, is fast in growth, easy to culture and short in fermentation period; the Staphylococcus sciuri CY1-78 strain is high in activity of producing the fibrinolysin, and the enzyme activity of shake flask fermentation can reach 1553 U / mL under the optimal condition; and the separation and purification process of the fibrinolysin is simple, and after salting-out and dialysis desalination, the activity of the freeze-dried powder of the fibrinolysin reaches 4030 U / mg.

Description

[0001] (1) Technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a staphylococcus squirrel (Staphylococcus sciuri) and its application in fermenting and preparing fibrinolytic enzyme. [0003] (2) Background technology [0004] Thrombotic disease seriously endangers human health. It is characterized by the formation of thrombus in blood vessels by formed components in blood, causing partial or complete blockage of blood vessels, thereby causing hypoxia, ischemia and necrosis of corresponding tissues or organs. Common thrombotic diseases mainly include myocardial infarction, angina pectoris, cerebral thrombosis, stroke, and pulmonary infarction. Thrombotic diseases not only have a high mortality rate but also a high disability rate, and the incidence rate is much higher than that of cancer. With the change of people's eating habits and the extension of life expectancy, the incidence of thrombotic diseases is also increasing y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/52C12R1/44
CPCC12N9/52C12R2001/44C12N1/205
Inventor 陈虹张建芬谢广发陆胤孙亚媛
Owner ZHEJIANG SHUREN UNIV
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