Genetically engineered medicine for treating brain injury and preparation method thereof

Abstract

The invention provides a genetically engineered drug for treating brain injury and a preparation method thereof. The preparation method specifically comprises the following operation steps: S1, sampling, extracting and mixing biological enzyme; s2, transferring genes, and culturing mother cells; and S3, preliminary separation and in-vivo experiment. Brain injury often causes various pathological changes such as encephaledema, nerve cell death and neuroinflammation, and the pathological changes are always carried out simultaneously. Therefore, the complex pathological processes are only symptomatic treatment at present, and after main death related proteins, especially caspase-8 and representative inflammatory factors after injury are detected, the genetic engineering medicine is found to be capable of blocking nerve cell death after brain injury and effectively inhibiting inflammatory response, so that the nerve cell death after brain injury can be inhibited. And therefore, neuronal degenerative change caused by brain injury and formation of encephaledema can be effectively inhibited.

Description

A genetically engineered drug for treating brain damage and its preparation method

[0001] This application claims priority to the invention patent application filed with the China Patent Office on March 1, 2022, with application number 2022101940447 and invention name “A genetically engineered drug for treating brain injury and its preparation method”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering drug for treating brain damage and a preparation method thereof. Background Art

[0003] Brain injury refers to the destruction and death of glial cells (including neurons and glial cells) in brain tissue caused by various factors, leading to neurological dysfunction. Due to the non-regenerative nature of neurons, brain injury is often a high-risk factor for various neuropsychiatric and neurodegenerative diseases. Furthermore, brain injury has a high incidence, mortality, and disability rate, severely impacting patients' quality of life and placing a significant burden on society. In the United States, approximately 2.6 million cases of brain injury occur annually. Depending on the cause, brain injury can be categorized as traumatic brain injury (TBI) or acquired brain injury (ABI). Because the severity of brain injury varies greatly, and damage to different brain regions can cause different symptoms—for example, focal brain injury often causes symptoms related to abnormalities in movement, sensation, speech, vision, and hearing, while diffuse brain injury often affects memory and sleep or leads to confusion and coma—there is currently no clear, unified approach to treating patients with brain injury.

[0004] Currently, the treatment for brain injury is mainly symptomatic treatment: analgesics can be given for headaches, but anesthetics or morphine-like drugs should not be used to avoid addiction, such as levorotatory cephalosporin, enteric-coated aspirin, naproxen, ibuprofen, etc.; diphenhydramine, chlorobutanol, etc. can be given for dizziness; oryzanol, promethazine, γ-aminobutyric acid (γ-aminobutyric acid), methylphenidate (methylphenidate), atropine (atropine sulfate), scopolamine, etc. can be given for autonomic dysfunction; excited patients can be given perphenazine, diazepam (Valium), oxazepam (norhydroxydiazepam), etc.; depressed patients can be given glutamate and γ-aminobutyric acid.

[0005] The present invention takes brain trauma (but not limited to brain trauma) as an example of brain injury, and explains the role of the invention from the aspects of nerve cell death, inflammation, motor function, learning and memory ability, and autonomous exploration ability after brain trauma.

[0006] Summary of the Invention

[0007] The object of the present invention is to provide a lignin-phenolic resin molding compound to solve the problems raised in the above background technology.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a genetically engineered drug for treating brain damage and a preparation method thereof, specifically according to the following operating steps:

[0009] S1: Bio-enzyme sampling, extraction and mixing

[0010] (a) extracting and expressing glycosylated monomeric metalloproteinases from mammals;

[0011] (b) activating the glycosylated monomeric metalloprotease in a reducing solution with a mixing ratio of 1:2-1:3 to optimize its activity, and then lysing it with protein water;

[0012] S2: Gene transfer and mother cell cultivation

[0013] The 978-bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to generate pAAV9 SP146:ACP5 as an expression cassette;

[0014] S3: Preliminary separation and in vivo experiments

[0015] (a) The transfection products were routinely coated on LB plates containing 80-100 mg / L Amp. The constructed plasmids were confirmed by sequencing and amplified for production.

[0016] (b) The transfected product is centrifuged at 10,000-25,000 rpm for 10-30 minutes at 0-8°C to obtain the supernatant and precipitate. The supernatant is subjected to chromatographic separation and purification procedures.

[0017] (c) Take viruses and conduct in vitro experiments

[0018] By injecting recombinant adeno-associated virus (rAAV) virus into mice, and injecting the above-synthesized glycosylated monomeric metalloproteinase;

[0019] (d) Record the biological characteristics of the mice.

[0020] Preferably, the coding sequence of the virus is:

[0021] Gene ID:54

[0022] acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase.

[0023] The technical effects and advantages of the present invention are as follows:

[0024] In the present invention, brain injury often leads to multiple pathological changes such as brain edema, neuronal cell death and neuroinflammation, and these pathological changes are often carried out simultaneously. Therefore, at present, only symptomatic treatment is used for these complex pathological processes. After detecting the main death-related proteins (especially caspase-8) and representative inflammatory factors after injury, it is found that the present invention can block the occurrence of neuronal cell death after brain injury, and can effectively suppress the inflammatory response at the same time. Based on this, the present invention can also effectively suppress the formation of neuronal degeneration and brain edema caused by brain injury.

[0025] Reference numerals

[0026] Figure 1 is based on the ring segment in the present invention;

[0027] FIG2 is an image of a gene fragment in the present invention;

[0028] FIG3 is a diagram showing the results of a Wire-grip experiment in the present invention;

[0029] FIG4 is a diagram showing the experimental results of the Escapegoat latency in the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] Example 1

[0032] A genetically engineered drug for treating brain damage and a preparation method thereof, specifically comprising the following steps:

[0033] S1: Bio-enzyme sampling, extraction and mixing

[0034] (a) extracting and expressing glycosylated monomeric metalloproteinases from mammals;

[0035] (b) activating the glycosylated monomeric metalloprotease in a reducing solution at a mixing ratio of 1:3 to optimize its activity, and then lysing it with protein water;

[0036] S2: Gene transfer and mother cell cultivation

[0037] The 978-bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to generate pAAV9 SP146:ACP5 as an expression cassette;

[0038] S3: Preliminary separation and in vivo experiments

[0039] (a) The transfection products were routinely coated on LB plates containing 100 mg / L Amp. The constructed plasmids were confirmed by sequencing and amplified for production.

[0040] (b) The transfected product was centrifuged at 25,000 rpm for 30 min at 8°C to obtain the supernatant and precipitate. The supernatant was subjected to chromatographic separation and purification.

[0041] (c) Take viruses and conduct in vitro experiments

[0042] The recombinant adeno-associated virus (rAAV) was injected into mice, and the above-synthesized glycosylated monomeric metalloprotease was injected. The coding sequence of the virus is:

[0043] Gene ID:54

[0044] acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase;

[0045] (d) Record the biological characteristics of the mice.

[0046] Example 2

[0047] A genetically engineered drug for treating brain damage and a preparation method thereof, specifically comprising the following steps:

[0048] S1: Bio-enzyme sampling, extraction and mixing

[0049] (a) extracting and expressing glycosylated monomeric metalloproteinases from mammals;

[0050] (b) activating the glycosylated monomeric metalloprotease in a reducing solution at a mixing ratio of 1:2 to optimize its activity, and then lysing it with protein water;

[0051] S2: Gene transfer and mother cell cultivation

[0052] The 978-bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to generate pAAV9 SP146:ACP5 as an expression cassette;

[0053] S3: Preliminary separation and in vivo experiments

[0054] (a) The transfection products were routinely coated on LB plates containing 80 mg / L Amp. The constructed plasmids were confirmed by sequencing and amplified for production.

[0055] (b) The transfected product was centrifuged at 10,000 rpm for 10 min at 0°C to obtain the supernatant and precipitate. The supernatant was subjected to chromatographic separation and purification.

[0056] (c) Take viruses and conduct in vitro experiments

[0057] The recombinant adeno-associated virus (rAAV) was injected into mice, and the above-synthesized glycosylated monomeric metalloprotease was injected. The coding sequence of the virus is:

[0058] Gene ID:54

[0059] acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase;

[0060] (d) Record the biological characteristics of the mice.

[0061] Example 3

[0062] A genetically engineered drug for treating brain damage and a preparation method thereof, specifically comprising the following steps:

[0063] S1: Bio-enzyme sampling, extraction and mixing

[0064] (a) extracting and expressing glycosylated monomeric metalloproteinases from mammals;

[0065] (b) activating the glycosylated monomeric metalloprotease in a reducing solution at a mixing ratio of 1:2.5 to optimize its activity, and then lysing it with protein water;

[0066] S2: Gene transfer and mother cell cultivation

[0067] The 978-bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to generate pAAV9 SP146:ACP5 as an expression cassette;

[0068] S3: Preliminary separation and in vivo experiments

[0069] (a) The transfection products were routinely coated on LB plates containing 85 mg / L Amp. The constructed plasmids were confirmed by sequencing and amplified for production.

[0070] (b) The transfected product was centrifuged at 23,000 rpm for 27 min at 5°C to obtain the supernatant and precipitate. The supernatant was subjected to chromatographic separation and purification.

[0071] (c) Take viruses and conduct in vitro experiments

[0072] The recombinant adeno-associated virus (rAAV) was injected into mice, and the above-synthesized glycosylated monomeric metalloprotease was injected. The coding sequence of the virus is:

[0073] Gene ID:54

[0074] acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase;

[0075] (d) Record the biological characteristics of the mice.

[0076] Tartrate-resistant acid phosphatase (TRAP or TRAPase), also known as ACP5, is a glycosylated monomeric metalloproteinase expressed in mammals. It has a molecular weight of approximately 35 kDa, an alkaline isoelectric point (7.6–9.5), and optimal activity under acidic conditions. TRAP is synthesized as a latent zymogen and activated by proteolytic cleavage and reduction. It differs from other mammalian acid phosphatases by its inhibition by tartrate and its molecular weight.

[0077] Mammalian TRAP is encoded by a gene located on chromosome 19 (19p13.2–13.3) in humans and chromosome 9 in mice. As expected from protein sequencing, TRAP DNA is highly conserved throughout the mammalian class. The human, mouse, and porcine TRAP genes all contain five exons, with an ATG codon at the beginning of exon 2; exon 1 is noncoding. Within exon 1, there are three distinct "tissue-specific" promoters: 1A, 1B, and 1C. This allows for tight control of TRAP expression. Transcribed from this gene is a 1.5 kb mRNA with a 969–975 bp open reading frame (ORF), encoding a protein of 323–325 amino acids. In rats, the ORF is 981 bp long and encodes a 327-amino acid protein. TRAP is translated as a single polypeptide. TRAP gene transcription is regulated by microphthalmia-associated transcription factors.

[0078] Recombinant adeno-associated virus (rAAV) is considered one of the most promising vectors for genetic research and gene therapy due to its minimal immunogenicity, high safety, broad host cell range (infecting both dividing and non-dividing cells), robust spread, and prolonged gene expression in vivo. The key structure of the rAAV pseudotype 9 packaged genome is as follows: the human ACP5 gene is driven by a synthetic macrophage-specific promoter, SP146.

[0079] Virus-related plasmid: pAAV9-SP146-ACP5.

[0080] Construction method: The CDS sequence of ACP5 was synthesized by Genescript (Suzhou, China). The Infusion system was used to construct the vector according to the instruction manual (Clontech). The vector frame and insert sequence were mixed in a 1 / 1 ratio in a total volume of 5 μL and the same volume was added to the GeneArt TM The enzyme mixture was seamlessly connected and mixed thoroughly with a pipette. The test tube was incubated at 50°C to complete the connection and heat-shocked into Stable3 competent cells (E. coli). The 978bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to produce pAAV9SP146:ACP5 as an expression frame. The transfection product was routinely coated in an LB plate containing 100mg / LAmp. The constructed plasmid was confirmed by sequencing and amplified for production. Virus production was completed by Genescript (Suzhou, China), and downstream AAV was purified using the AAVpro purification kit (TakaraBioUSA).

[0081] Experimental data:

[0082] Provide further data proof of the effects (advantages) achieved by this product or method.

[0083] Mouse Model: The controlled cortical impact (CCI) is a commonly used instrument for establishing rodent models of traumatic brain injury (TBI). This animal model is recognized as one of the most effective models for simulating brain injury. Male, 6-8-week-old C57BL6 / J mice were used as research subjects. A TBI model was established in mice using the CCI instrument with an impact depth of 1 mm. Following injury, 2 μl of AAV-ACP5 and AAV-NC (negative control) were administered via contralateral intracerebroventricular injection. Changes in cell death-related proteins and inflammation-related proteins were measured in the injured side of the brain tissue 1 day post-injury (dpi). Behavioral changes were also assessed 1–20 days after injury.

[0084] result:

[0085] As shown in Figure 2-4

[0086] Western blotting was used to detect changes in caspase family proteins in the injured brain tissue. After AAV-ACP5 treatment, the expression levels of caspase family proteins in the injured brain tissue of mice were significantly reduced, indicating that AAV-ACP5 treatment can alleviate neuronal cell death caused by brain trauma.

[0087] The protein changes of inflammatory factors in the injured side brain tissue were detected by immunoblotting: After AAV-ACP5 treatment, the expression level of inflammatory factors in the injured side brain tissue of mice was significantly reduced, that is, AAV-ACP5 treatment can alleviate neuroinflammation in brain tissue caused by brain trauma.

[0088] Through relevant behavioral tests, the changes in motor function, learning and memory abilities of mice after TBI were detected: mice treated with AAV-ACP5 showed significantly stronger motor ability, better learning and memory abilities, that is, AAV-ACP5 treatment can promote the recovery of motor function, learning and memory abilities after TBI.

[0089] In the present invention, brain injury often leads to multiple pathological changes such as brain edema, neuronal cell death and neuroinflammation, and these pathological changes are often carried out simultaneously. Therefore, at present, only symptomatic treatment is used for these complex pathological processes. After detecting the main death-related proteins (especially caspase-8) and representative inflammatory factors after injury, it is found that the present invention can block the occurrence of neuronal cell death after brain injury, and can effectively suppress the inflammatory response at the same time. Based on this, the present invention can also effectively suppress the formation of neuronal degeneration and brain edema caused by brain injury.

Claims

1. A genetically engineered drug for treating brain damage and a method for preparing the same, characterized in that: Follow these steps: S1: Bio-enzyme sampling, extraction and mixing (a) extracting and expressing glycosylated monomeric metalloproteinases from mammals; (b) activating the glycosylated monomeric metalloprotease in a reducing solution with a mixing ratio of 1:2-1:3 to optimize its activity, and then lysing it with protein water; S2: Gene transfer and cultivation of mother cells The 978-bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to generate pAAV9 SP146:ACP5 as an expression cassette; S3: Preliminary separation and in vivo experiments (a) The transfection products were routinely coated on LB plates containing 80-100 mg / L Amp. The constructed plasmids were confirmed by sequencing and amplified for production. (b) The transfected product is centrifuged at 10,000-25,000 rpm for 10-30 minutes at 0-8°C to obtain the supernatant and precipitate. The supernatant is subjected to chromatographic separation and purification procedures. (c) Take viruses and conduct in vitro experiments By injecting recombinant adeno-associated virus (rAAV) virus into mice, and injecting the above-synthesized glycosylated monomeric metalloproteinase; (d) Record the biological characteristics of the mice.

2. A genetically engineered drug for treating brain damage and a preparation method thereof according to claim 1, characterized in that: The coding sequence of the virus is: Gene ID:54 acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase.

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