Fusion protein of cytoglobin and sipunculus nudus plasmin

A technology of fusion protein and gridworm, which is applied in the biological field to achieve high biological activity and good thrombolytic effect

Active Publication Date: 2019-01-25
许瑞安
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are still few marine cardiovascular drugs that can be applied clinically, and there are few reports on the gene cloning and expression of plasmin from marine organisms, this is a promising field worth exploring.

Method used

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  • Fusion protein of cytoglobin and sipunculus nudus plasmin
  • Fusion protein of cytoglobin and sipunculus nudus plasmin
  • Fusion protein of cytoglobin and sipunculus nudus plasmin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Construction of embodiment 1 recombinant plasmid pPIC9-CYGB-PL

[0054] (1) The plasmid pET22b-CYGB-PL was used as a template to PCR amplify the CYGB-PL sequence, and the system was as follows:

[0055]

[0056] Reaction conditions: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 s, annealing at 53°C for 30 s, extension at 72°C for 1 min and 10 sec, cycled 5 times; final 10 min at 72°C, and storage at 4°C. Mix 50 μL of the PCR product with 10 μL 6×loading buffer evenly; electrophoresis at 90V for 25 minutes, take out the gel after electrophoresis, observe and take pictures under the gel imaging analyzer, and recover the target fragment from the gel. glue recycling;

[0057] (2) Double digestion of pPIC9 plasmid and gel-recovered CYGB-PL DNA fragment

[0058] The double enzyme digestion system is as follows:

[0059]

[0060] Reaction conditions: 37°C water bath overnight (12h), after the reaction is completed, the digested product is recovered b...

Embodiment 2

[0065] Colony PCR verification of embodiment 2 recombinant pPIC9-CYGB-PL

[0066] The identification system is as follows:

[0067]

[0068] Reaction conditions: pre-denaturation at 94°C, 4min cycles of denaturation at 94°C for 45s, annealing at 52°C for 30s, extension at 72°C for 1min10s, and 25 cycles; finally, 72°C, 10min, and storage at 4°C. Take 5 μL of the PCR product and 1 μL of 6×loading buffer, mix and load the sample, electrophoresis at 90V for 25 minutes, take out the gel after electrophoresis, observe and take pictures under the gel imaging analyzer.

Embodiment 3

[0069] Example 3 Double enzyme digestion and PCR verification of recombinant plasmid pPIC9-CYGB-PL

[0070] Inoculate a single colony with a positive identification result into 5 mL of LB medium containing Amp, culture on a shaker at 250 rpm at 37°C for about 13 hours, and then extract the plasmid. The plasmid double enzyme digestion system is as follows:

[0071]

[0072] Reaction conditions: 37°C water bath for 7 hours, then perform gel electrophoresis and take pictures to record the results.

[0073] The plasmid PCR system is as follows:

[0074]

[0075] The single colonies with positive results of plasmid double enzyme digestion and plasmid PCR identification were sent to Nanjing GenScript Co., Ltd. for sequence determination.

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Abstract

The invention discloses fusion protein of cytoglobin and sipunculus nudus plasmin, and further provides a preparation method for the fusion protein and related bacterial strain construction. The method comprises the following steps that bacterial strain culture and amplification are carried out, supernatant is separated, sulfuric acid precipitating is carried out, Sephadex G-25 desalting is carried out, and DEAE step-by-step elution and purification are carried out so as to obtain the fusion protein. According to the fusion protein, the optimum pH range is within 5.0-9.0 at 40 DEG C or below,the enzyme activity keeps relatively stable, and the influence of PMSF and Cu<2+> on the activity of fusion protease is relatively large. The fusion protein has the characteristic of targeted degradation of fibrinogen, and the degradation sequence is firstly an alpha chain, secondly a beta chain and finally a gamma chain. The fusion protein has a better thrombolytic effect on urokinase in a thrombus experiment.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a fusion protein of cytoglobin and plasminus fibrinolytic enzyme, their preparation method and their efficacy. Background technique [0002] Thromboembolic disease is a common cardiovascular and cerebrovascular disease, and its incidence rate ranks first among various diseases. The global potential market for thrombolytics is about $2 billion per year. Marine active substances have gradually become a research hotspot in recent years because of their novel structure and high pharmacological activity. More and more fibrinolytic active substances have been identified from the ocean, many of which can not only activate plasminogen but also directly dissolve fibrin. During clinical application, if plasminogen is activated with an external plasmin activator, and exogenous enzymes that can directly degrade fibrin are used, the plasma fibrinolytic activity can be additi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/81C12N1/19A61K38/48A61K47/42A61P7/02C12R1/84
CPCA61P7/02C12N9/6435C12N15/815C07K14/47C07K2319/00A61K38/00
Inventor 许瑞安李招发陈磊肖玲慧马国兴崔秀灵
Owner 许瑞安
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