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Pgk1 protein, recombinant plasmid for expressing Pgk1 protein, recombinant probiotic for expressing Pgk1 protein and application

A technology of recombinant plasmids and probiotics, applied in the direction of peptide/protein components, applications, recombinant DNA technology, etc., to improve neurological behavior, significant therapeutic effect, and reduce the volume of cerebral infarction

Pending Publication Date: 2021-11-05
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no literature disclosing that Pgk1 can be applied to the treatment of cardiovascular and cerebrovascular diseases and metabolic diseases.

Method used

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  • Pgk1 protein, recombinant plasmid for expressing Pgk1 protein, recombinant probiotic for expressing Pgk1 protein and application
  • Pgk1 protein, recombinant plasmid for expressing Pgk1 protein, recombinant probiotic for expressing Pgk1 protein and application
  • Pgk1 protein, recombinant plasmid for expressing Pgk1 protein, recombinant probiotic for expressing Pgk1 protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of recombinant plasmids expressing Pgk1 and recombinant probiotics expressing Pgk1

[0060] 1. Primer Synthesis

[0061] According to the nucleotide sequence of the gene Pgk1 (shown in SEQ ID NO.2), compare it with the pET-28a plasmid map, design specific primers, and insert restriction endonuclease sites BamHI and EcoRI into the primers, specific information See Table-1. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.; the primers were dissolved in sterile deionized water to make a concentration of 10 μmol / L.

[0062] Table 1 Primer information and synthesis

[0063]

[0064] 2. Construction of recombinant plasmids

[0065] The pET-28a-Pgk1 plasmid was synthesized by Jinweizhi Biotechnology Co., Ltd.

[0066] 3. Transform Escherichia coli DH5α bacteria with recombinant plasmid

[0067] Take out the DH5α competent cells from the -80℃ refrigerator, and place them on the ice box for 10-20 minutes to melt them; take out...

Embodiment 2

[0079] Example 2 Therapeutic Effect of Recombinant Probiotics Expressing Pgk1 on Ischemic Stroke Rats

[0080] 1. Preparation and administration time of MCAO model:

[0081] SPF grade healthy male SD rats, weighing 180-220g, were randomly divided into 7 groups after one week of adaptive feeding, and the grouping and dosage were as follows:

[0082] Sham operation group (SHAM, orally administered β-lactose solution of equal volume 11.1mg / ml);

[0083] MCAO ischemia-reperfusion model group (IR, orally administered an equal volume of 0.9% normal saline);

[0084] MCAO model+lactose group (IR+lactose, oral administration of equal volume of 11.1mg / ml β-lactose solution);

[0085] MCAO model + positive drug group (NBP, oral administration of butylphthalide 20mg / kg / day);

[0086] MCAO model + E.coli Nissle 1917 group: Escherichia coli (Escherichia coli) Nissle 1917 original bacteria of 2.5mL orally administered;

[0087] MCAO model+pET-28a-EGFP group: intragastric administration ...

Embodiment 3

[0102] Example 3 The therapeutic effect of recombinant probiotics expressing Pgk1 on diabetic mice

[0103] 1. Establishment of C57BL / 6N diabetic mouse model

[0104] SPF grade healthy male C57BL / 6N mice, weighing 18-22g.

[0105] Sixty C57BL / 6N mice were randomly divided into three groups:

[0106] Common diet group: SD group, 8 C57BL / 6N mice. After feeding with common feed for three weeks, intraperitoneally inject citrate buffer solution (the injection dose is the same as the following STZ solution), fasting and water for 12 hours before injecting citrate buffer solution, inject according to the same time period 9:00am-10:00am, Inject once a day for five consecutive days, and feed after 2 hours of citric acid buffer solution injection. Then feed with common feed for another three weeks, fasting without food and water for 10 hours, and detect fasting blood glucose of mice between 7:30pm-9:00pm.

[0107]Type 2 diabetes model group: high-fat diet + streptozotocin group, HFD...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a Pgk1 protein, a recombinant plasmid for expressing the Pgk1 protein, a recombinant probiotic for expressing the Pgk1 protein and application. According to the invention, firstly, inserting a Pgk1 gene into an expression vector pET-28a to obtain a recombinant plasmid for expressing Pgk1; and secondly, introducing the recombinant plasmid into an Escherichia coli Nissle 1917 tolerant bacterium, so that a functional recombinant probiotic is obtained. The recombinant probiotics can effectively inhibit cell death, improve the cerebral infarction volume and reduce blood sugar while efficiently expressing the Pgk1 protein, has a remarkable curative effect on cerebrovascular diseases and diabetes mellitus, has higher curative effect than that of clinical drugs, and has a good clinical application prospect.

Description

technical field [0001] The present invention relates to the field of biopharmaceuticals, in particular to a Pgk1 protein, a recombinant plasmid expressing the Pgk1 protein, a new application of the recombinant probiotic Pgk1 protein expressing the Pgk1 protein, a recombinant probiotic expressing the Pgk1, and a preparation for treating and / or preventing heart disease. Use of drugs for cerebrovascular and metabolic diseases. [0002] technical background [0003] Cerebrovascular disease refers to various diseases of brain vessels, including atherosclerosis, thrombosis, stenosis, occlusion, cerebral arteritis, cerebral artery injury, cerebral aneurysm, intracranial vascular malformation, cerebral arteriovenous fistula etc., whose common feature is to cause ischemia or hemorrhagic accident of brain tissue. Typical cerebrovascular diseases mainly include cerebral edema, cerebral thrombosis, cerebral ischemia, cerebral infarction, and stroke. Cerebral thrombosis is based on cere...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/53C12N1/21A61K38/45A61K35/741A61K48/00A61K9/00A61P9/00A61P3/00A61P9/10A61P9/04A61P7/10A61P25/00A61P7/02A61P3/10A61P39/02A61P3/08A61P19/06A61P3/02A61P19/10C12R1/19
CPCC12N15/70C12N9/1217A61K38/45A61K35/741A61K48/005A61K9/0053A61P9/00A61P3/00A61P9/10A61P9/04A61P7/10A61P25/00A61P7/02A61P3/10A61P39/02A61P3/08A61P19/06A61P3/02A61P19/10C12Y207/02003Y02A50/30
Inventor 陈新平刘菁菁周静赵文阳李纯吴桐雨
Owner LANZHOU UNIVERSITY
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