A polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid complex and application thereof
Patent Information
- Application Number
- CN202410631445.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-21
- Publication Date
- 2026-10-23
AI Technical Summary
[0003]目前，多肽-脂质体与人血管内皮生长因子(VEGF)基因重组质粒复合物在制备时发现，目前制备的步骤较为繁杂，导致制备的质粒复合物制备的效果较差，进而导致其应用效果较差，不适合大面积的推广与应用
[0025]本发明通过在制备前对原料进行筛选处理，可以精准的获得较好的原理，提高了复合物制备的效果，且在制备的过程中加入辅助剂，可以提高复合物的稳定性、降低细胞毒性，并增强基因传递效果，同时通过对制备的复合物进行检测评估，可以精准了解复合物的功能，以确保复合物的功能和可靠性，进而提高了复合物的应用效果。
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Figure CN122954986A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically a complex of a polypeptide-liposome and a recombinant plasmid of human vascular endothelial growth factor gene and its application. Background Technology
[0002] The peptide-liposome-human vascular endothelial growth factor (VEGF) gene recombinant plasmid complex refers to the combination of VEGF gene recombinant plasmid with a specific peptide vector (such as a peptide delivery vector) and the formation of a complex by encapsulating it in a liposome. This complex can be used in fields such as non-viral mediated gene delivery and gene therapy.
[0003] Currently, the preparation of peptide-liposome and human vascular endothelial growth factor (VEGF) gene recombinant plasmid complexes is quite complicated, resulting in poor plasmid complex preparation quality and consequently poor application performance, making it unsuitable for large-scale promotion and application.
[0004] To address the problems raised in the background art, those skilled in the art have proposed a polypeptide-liposome-human vascular endothelial growth factor gene recombinant plasmid complex and its application. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a complex of a polypeptide-liposome and a recombinant plasmid of human vascular endothelial growth factor gene and its application, thereby resolving the issues existing in the prior art.
[0006] A polypeptide-liposome-human vascular endothelial growth factor gene recombinant plasmid complex, the plasmid complex comprising the following ingredients by weight percentage: 10-15% polypeptide carrier, 60-80% liposome, 15-25% gene recombinant plasmid and adjuvant.
[0007] Preferably, the polypeptide carrier is a vector for targeting cells or delivering genes within cells; wherein the polypeptide is synthesized using chemical synthesis or genetic engineering techniques, and the sequence of the polypeptide should contain affinity groups that interact with liposomes, such as hydrophilic or hydrophobic amino acid sequences, and its amino acid residue sequence and primary structure are GNOSSNFGPMKGGNFGGRSSGPYGGGGOYFAKPRNOGGYPKKKRKV.
[0008] The sequence GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY is derived from the nuclear localization signal sequence of the intranuclear heterologous riboprotein A1 protein; the liposomes are prepared by membrane dissolution, membrane fusion, or reverse emulsification, and are typically composed of phospholipids, cholesterol, and other functional components; the recombinant plasmid is a vector carrying the target gene; and the adjuvant is at least one of a polymer, a protein, and polyethyleneimine (PEI).
[0009] Preferably, the PKKKRKV sequence is derived from the nuclear localization signal sequence of the T antibody of simian vacuolating virus 40; the polypeptide is bound to the human vascular endothelial growth factor gene recombinant plasmid and liposome in a non-covalent manner, wherein the liposome is an artificially synthesized lipid substance that can encapsulate the gene recombinant plasmid or DNA and help the recombinant plasmid or DNA cross the membrane structure of mammalian cells.
[0010] A method for preparing a complex of a polypeptide-liposome and a recombinant plasmid containing the human vascular endothelial growth factor gene includes the following steps:
[0011] S1. Material selection: First, select appropriate amounts of polypeptide vectors, liposomes, recombinant plasmids and auxiliary agents. Then, screen the polypeptide vectors, liposomes and recombinant plasmids to obtain polypeptide vectors, liposomes and recombinant plasmids in good condition and with uniform morphology.
[0012] S2. Preparation of peptide-liposome complex: The synthesized peptide is then combined with the prepared liposome. This can be achieved by eutectic method, membrane dissolution method or ultrasonic treatment method, and a small amount of auxiliary agent is added during the preparation of the complex.
[0013] S3. Preparation of VEGF gene recombinant plasmid: Then, a plasmid containing the VEGF gene is prepared using genetic engineering technology. That is, the VEGF gene sequence can be amplified by PCR and inserted into the vector plasmid. Then, the plasmid is replicated and amplified to finally obtain a recombinant plasmid containing the VEGF gene. A small amount of auxiliary agent is added during the preparation of the complex.
[0014] S4. Preparation of peptide-liposome and VEGF gene recombinant plasmid complex: Finally, the peptide-liposome complex and VEGF gene recombinant plasmid are compounded. The peptide-liposome complex and VEGF gene recombinant plasmid can interact through methods such as co-melting or co-precipitation to form a stable complex. During the preparation of the complex, the remaining auxiliary agents are added.
[0015] S5. Obtain the complex of polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid.
[0016] S6. Validation and Evaluation: Finally, the complex of peptide-liposome and human vascular endothelial growth factor gene recombinant plasmid was tested and evaluated.
[0017] Preferably, a stirring rod is used for stirring and mixing during the preparation of the composite.
[0018] Preferably, in step S2, during the compounding process, the polypeptide binds to the liposome through its affinity group.
[0019] Preferably, in step S6, the detection and evaluation process includes the following steps:
[0020] S61. First, dynamic light scattering technology can be used to determine the particle size and morphology of the complex;
[0021] S62. Next, the gel migration assay can be used to detect the interaction between peptides and recombinant gene plasmids. The binding ability of peptides and recombinant gene plasmids can be evaluated by observing the migration of samples on the gel or the expression of proteins.
[0022] S63. Finally, cell experiments and in vivo experiments can be conducted to evaluate the function and effect of peptide-liposomes and gene recombinant plasmids; for example, cell transfection methods can be used to introduce the complex into cells and observe its effect on the expression of intracellular vascular endothelial growth factor gene and cell proliferation capacity.
[0023] The application of a polypeptide-liposome-human vascular endothelial growth factor gene recombinant plasmid complex prepared by a method for the treatment of ischemic peripheral vascular disease and ischemic coronary heart disease in gene therapy drugs.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] This invention achieves better results by screening raw materials before preparation, thus improving the preparation effect of the complex. Furthermore, the addition of auxiliary agents during the preparation process can improve the stability of the complex, reduce cytotoxicity, and enhance gene delivery. At the same time, by testing and evaluating the prepared complex, the function of the complex can be accurately understood to ensure its functionality and reliability, thereby improving the application effect of the complex. Attached Figure Description
[0026] Figure 1 This is a flowchart of the preparation method of the polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid complex of the present invention. Detailed Implementation
[0027] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.
[0028] As attached Figure 1 As shown:
[0029] Example: The present invention provides a polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid complex, the plasmid complex comprising the following raw materials by weight percentage: 10-15% polypeptide carrier, 60-80% liposome, 15-25% gene recombinant plasmid and auxiliary agent.
[0030] Specifically, the polypeptide carrier is a carrier used for targeted or intracellular gene delivery;
[0031] The liposomes are prepared by membrane dissolution, membrane fusion or reverse emulsification and are typically composed of phospholipids, cholesterol and other functional components.
[0032] The recombinant plasmid is a vector carrying the target gene;
[0033] The adjuvants include polymers, proteins, and polyethyleneimine (PEI).
[0034] A method for preparing a complex of a polypeptide-liposome and a recombinant plasmid containing the human vascular endothelial growth factor gene includes the following steps:
[0035] S1. Material selection: First, select appropriate amounts of polypeptide vectors, liposomes, recombinant plasmids and auxiliary agents. Then, screen the polypeptide vectors, liposomes and recombinant plasmids to obtain polypeptide vectors, liposomes and recombinant plasmids in good condition and with uniform morphology.
[0036] S2. Preparation of peptide-liposome complex: The synthesized peptide is then combined with the prepared liposome. During the preparation of the complex, a small amount of auxiliary agent is added, and the peptide binds to the liposome through its affinity group.
[0037] S3. Preparation of VEGF gene recombinant plasmid: Then, a plasmid containing the VEGF gene is prepared using genetic engineering technology. That is, the VEGF gene sequence can be amplified by PCR and inserted into the vector plasmid. Then, the plasmid is replicated and amplified to finally obtain a recombinant plasmid containing the VEGF gene. A small amount of auxiliary agent is added during the preparation of the complex.
[0038] S4. Preparation of peptide-liposome and VEGF gene recombinant plasmid complex: Finally, the peptide-liposome complex is compounded with the VEGF gene recombinant plasmid, and the remaining auxiliary agent is added during the complex preparation process.
[0039] S5. Obtain the complex of polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid.
[0040] S6. Detection and evaluation: Finally, the complex of polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid was detected and evaluated.
[0041] Specifically, the detection and evaluation process involves the following steps:
[0042] S61. First, dynamic light scattering technology can be used to determine the particle size and morphology of the complex;
[0043] S62. Next, the Gel migration assay can be used to detect the interaction between the peptide and the recombinant plasmid.
[0044] S63. Finally, cell experiments and in vivo experiments can be conducted to evaluate the function and effect of peptide-liposomes and gene recombinant plasmids.
[0045] As can be seen from the above, by screening and processing the raw materials before preparation, better principles can be obtained accurately, which improves the preparation effect of the complex. In addition, the addition of auxiliary agents during the preparation process can improve the stability of the complex, reduce cytotoxicity, and enhance gene delivery. At the same time, by testing and evaluating the prepared complex, the function of the complex can be accurately understood to ensure the function and reliability of the complex, thereby improving the application effect of the complex.
[0046] Application examples
[0047] 1. Application of polypeptide-liposome-human vascular endothelial growth factor gene recombinant plasmid complex in the treatment of ischemic peripheral vascular disease
[0048] One hundred patients with ischemic peripheral vascular disease were randomly divided into two groups. The experimental observation group was treated with the complex prepared in the example; the control group was treated with conventional treatment methods. The patients' conditions were observed after six months, and the results are shown in the table below:
[0049] Experimental observation group 89.4％ control group 76.4％
[0050] The table above shows that the total effective rate of the observation group was significantly better than that of the control group. Observing its therapeutic effect, it can be seen that the polypeptide-liposome complex with human vascular endothelial growth factor gene recombinant plasmid prepared in this embodiment can effectively treat ischemic peripheral vascular disease.
[0051] 2. Application of polypeptide-liposome-human vascular endothelial growth factor gene recombinant plasmid complex in the treatment of ischemic coronary heart disease
[0052] Sixty patients with ischemic coronary heart disease were randomly divided into two groups. The experimental observation group was treated with the complex prepared in the example; the control group was treated with conventional treatment methods. The patients' conditions were observed after six months, and the results are shown in the table below:
[0053] Experimental observation group 88.6％ control group 74.1％
[0054] The table above shows that the total effective rate of the observation group was significantly better than that of the control group. Observing its efficacy, it can be seen that the polypeptide-liposome complex with human vascular endothelial growth factor gene recombinant plasmid prepared in this example can effectively treat ischemic coronary heart disease.
[0055] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. A complex of a polypeptide-liposome and a recombinant plasmid containing the human vascular endothelial growth factor gene, characterized in that: The plasmid complex comprises the following ingredients by weight percentage: 10-15% polypeptide vector, 60-80% liposomes, 15-25% recombinant plasmid and auxiliary agent.
2. The polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid complex as described in claim 1, characterized in that: The polypeptide carrier is a carrier used for targeted or intracellular gene delivery; The liposomes are prepared by membrane dissolution, membrane fusion or reverse emulsification and are typically composed of phospholipids, cholesterol and other functional components. The recombinant plasmid is a vector carrying the target gene; The adjuvants include polymers, proteins, and polyethyleneimine (PEI).
3. The method for preparing a polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid complex as described in any one of claims 1-2, characterized in that: Includes the following steps: S1. Material selection: First, select appropriate amounts of polypeptide vectors, liposomes, recombinant plasmids and auxiliary agents. Then, screen the polypeptide vectors, liposomes and recombinant plasmids to obtain polypeptide vectors, liposomes and recombinant plasmids in good condition and with uniform morphology. S2. Preparation of peptide-liposome complex: The synthesized peptide is then combined with the prepared liposome, and a small amount of auxiliary agent is added during the preparation of the complex. S3. Preparation of VEGF gene recombinant plasmid: Then, a plasmid containing the VEGF gene is prepared using genetic engineering technology. That is, the VEGF gene sequence can be amplified by PCR and inserted into the vector plasmid. Then, the plasmid is replicated and amplified to finally obtain a recombinant plasmid containing the VEGF gene. A small amount of auxiliary agent is added during the preparation of the complex. S4. Preparation of peptide-liposome and VEGF gene recombinant plasmid complex: Finally, the peptide-liposome complex is compounded with the VEGF gene recombinant plasmid, and the remaining auxiliary agent is added during the complex preparation process. S5. Obtain the complex of polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid. S6. Detection and evaluation: Finally, the complex of polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid was detected and evaluated.
4. The method for preparing a polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid complex as described in claim 1, characterized in that: In step S2, during the compounding process, the polypeptide binds to the liposome through its affinity group.
5. The method for preparing a polypeptide-liposome and human vascular endothelial growth factor gene recombinant plasmid complex as described in claim 1, characterized in that: In step S6, the detection and evaluation process includes the following steps: S61. First, dynamic light scattering technology can be used to determine the particle size and morphology of the complex; S62. Next, the Gel migration assay can be used to detect the interaction between the peptide and the recombinant plasmid. S63. Finally, cell experiments and in vivo experiments can be conducted to evaluate the function and effect of peptide-liposomes and gene recombinant plasmids.
6. The application of the polypeptide-liposome-human vascular endothelial growth factor gene recombinant plasmid complex prepared by the method for preparing the polypeptide-liposome-human vascular endothelial growth factor gene recombinant plasmid complex according to any one of claims 3-5 in gene therapy drugs for treating ischemic peripheral vascular disease and ischemic coronary heart disease.