Gene vaccine for preventing and treating tumors and preparation method and application of gene vaccine

A genetic vaccine and gene technology, applied in the field of preparation and design of genetic vaccines, can solve the problems of low antigen presentation efficiency, complex vaccine components, poor integration, etc., to improve long-term effect and stability, and improve antigen presentation efficiency , the effect of reducing the free time

Active Publication Date: 2017-10-24
杭州贝罗康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is: the expression products of existing similar gene vaccines have short half-life in vivo, low antigen presentation efficiency, complex vaccine components, poor integration and poor synergy

Method used

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  • Gene vaccine for preventing and treating tumors and preparation method and application of gene vaccine
  • Gene vaccine for preventing and treating tumors and preparation method and application of gene vaccine
  • Gene vaccine for preventing and treating tumors and preparation method and application of gene vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1: Construction of vaccine plasmid pVAX1-AFP-linker-Fc-IRES-CSF2

[0069] Plasmids and strains

[0070] The recombinant plasmid H3088pAV-MCMV-AFP containing the AFP gene sequence was synthesized and constructed by Shanghai Yingjun Biotechnology Co., Ltd., and the GenBank sequence number of AFP is: NM_001134.2.

[0071] The cDNA clones containing pVAX1 eukaryotic expression vector and CSF2 were purchased from Changsha Yingrun Biotechnology Co., Ltd., and the GenBank sequence number of CSF2 is: BC113999.1.

[0072] The plasmid vector pIRES2-EGFP containing the IRES sequence was donated by the laboratory of Zhejiang University School of Medicine.

[0073] The Fc segment includes the CH2, CH3 and hinge regions of the human IgG1 heavy chain region. The sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the nucleotide sequence is NO: 2 in the sequence listing.

[0074] 1.1 PCR amplification of gene sequence AFP-linker.

[0075] The AFP gen...

Embodiment 2

[0120] Example 2: In vitro expression detection of vaccine plasmid pVAX1-AFP-Linker-Fc-IRES-CSF2

[0121] 2.1 Plasmid-transfected cells

[0122] Observe under a microscope, and transfect when HEK293 cells grow to more than 80%. Set up a group of negative control group and a group of plasmid transfection experiment group, wherein DNA of negative control group adopts pVAX1 empty plasmid, DNA of plasmid transfection experiment group adopts pVAX1-AFP-Linker-Fc-IRES-CSF2, uses PolyJet of SignaGen company TM DNA transfection reagent in vitro: The above-mentioned plasmid DNA is used to transfect HEK293 cells, and the specific steps are as follows (the following steps take the 6cm culture dish system as an example): 1. 60 minutes before transfection, use 2mL of 10% fetal bovine serum (Sijiqing) DMEM (Hyclone) medium was used to replace the cells; 2. Use DMEM serum-free high-glucose medium to dilute plasmid DNA and PolyJetTM transfection reagent, using DNA (μg): PolyJetTM (μL) ratio ...

Embodiment 3

[0130] Example 3. Vaccine inhibits the proliferation of HepG2 liver cancer cells

[0131] 3.1 Vaccine transfection inhibition test

[0132] 1.5 x 10 per well 3 A HepG2 cell was seeded in a 24-well plate and injected into 500 μL DMEM high-glucose medium (containing 10% FBS) for culture. After culturing for one day, transfection was carried out. The control group and the experimental group were set up in each group, and each group was set up with 3 replicate wells. The plasmid DNA in the control group was pVAX1 empty plasmid, and the plasmid DNA in the experimental group was constructed with pVAX1-AFP-Linker-Fc. -IRES-CSF2 via PolyJet from SignaGen TMDNA in vitro transfection reagent The above plasmid DNA was transfected into HepG2 cells. The specific steps were according to the instructions of the reagents. A complex of 5 g of plasmid DNA and 1 μL of liposomes was added to each well of a 96-well plate for transfection, and culture was continued for 96 h in a low serum state ...

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Abstract

The invention discloses a gene vaccine for preventing and treating tumors. The gene vaccine comprises a recombinant gene eukaryotic expression vector carrying a human AFP gene, a human IgG1 Fc fragment gene and a human CSF2 gene at the same time. The gene vaccine can be applied to gene immunotherapy and prevention of diseases, such as a liver cancer, and is capable of developing the immunomodulatory effect of cytokines and generating a specific immune response through targeting to the diseases, such as the liver cancer; and the number of components of the vaccine is also reduced by adopting a dual-gene co-expression strategy. The test result shows that the vaccine is capable of normally expressing a target gene in an in-vitro human cell line and capable of significantly inhibiting division and proliferation of liver cancer cells under an in-vitro condition.

Description

technical field [0001] The invention relates to a genetic vaccine, in particular to a design, preparation method and application of a genetic vaccine capable of activating specific body immune responses and having the effect of preventing and treating liver cancer and other cancers. Background technique [0002] Genetic vaccine is a new type of vaccine that appeared in the early 1990s. It refers to the form of vaccine prepared by using the gene itself that can express a specific antigen. The current genetic vaccine is mainly eukaryotic expression plasmid DNA containing the antigenic gene. Since the emergence of genetic vaccines, they have achieved rapid and considerable development, and their applications have expanded from infectious and preventive vaccines to non-infectious diseases and therapeutic vaccines, including diabetes and tumor treatment. Compared with traditional vaccines, genetic vaccines can be industrially produced on a large scale, and have many advantages su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/39A61P35/00C12N15/85
CPCA61K39/0011A61K39/39A61K2039/53A61K2039/55522C12N15/85C12N2800/107Y02A50/30
Inventor 尚云龙曹王丽罗伊·杜曼尼杨军方玲胡江宁王如伟陈哲浩吴华铃吴永江
Owner 杭州贝罗康生物技术有限公司
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