Recombinant adeno-associated virus vector carrying human papillomavirus type 16 multi-point mutant e7mm antigen gene and its construction method and application

A human papilloma virus, hpv-16e7mm technology, applied in the application field of preparing anti-HPV-16 infection and related disease treatment drugs, and can solve problems such as safety risks

Active Publication Date: 2018-02-02
GUANGDONG TOPHEALTH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there are certain risks in terms of safety in stimulating immune responses with wild-type HPV-16 E7 antigen in vivo and in vitro

Method used

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  • Recombinant adeno-associated virus vector carrying human papillomavirus type 16 multi-point mutant e7mm antigen gene and its construction method and application
  • Recombinant adeno-associated virus vector carrying human papillomavirus type 16 multi-point mutant e7mm antigen gene and its construction method and application
  • Recombinant adeno-associated virus vector carrying human papillomavirus type 16 multi-point mutant e7mm antigen gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1, Construction of recombinant adeno-associated virus vectors AAV / HPV-16 E7 and AAV / HPV-16 E7 mm

[0073] 1. Materials and their sources:

[0074] A. Four kinds of adeno-associated virus (AAV) vectors: pAAV / p5 with AAV p5 promoter, pAAV / CMVp with macrophage virus (CMV) promoter, pAAV / SV40p with SV40 virus early promoter and pAAV / β-actinp with the human β-actin promoter. Known adeno-associated virus vectors have a p5 promoter. In order to increase the transcription level of the target gene, the p5 promoter in the recombinant adeno-associated virus vector can be replaced with a macrophage virus (cytomegalovirus, CMV) promoter, beta actin The promoter and one of the SV40 viral promoters, the four AAV vectors are only different in the promoter, and the rest of the gene structure is exactly the same, that is, it has the complete repeat terminal fragment (TR) sequence at both ends of the AAV type 2, and at both ends of the TR There are 9 nucleotide fragments (CTGCG...

Embodiment 2

[0089] Embodiment 2, preparation of recombinant adeno-associated virus (rAAV) and virus titer determination

[0090] Materials and their sources:

[0091] A. Carrying HPV-16 E7 constructed in Example 1 mm and HPV-16 E7 antigen gene recombinant adeno-associated virus vector (AAV / HPV-16 E7 mm and AAV / HPV-16 E7).

[0092] B. Auxiliary plasmid pHelper containing the Rep gene and Cap gene of AAV: successfully constructed by the inventors of this patent application, Liu Yong et al. (Liu, Y., Santin AD., Mane M., Chiriva-Internati, M., Parham GP ., Ravaggi, A., and Hermonat, P.L. Transduction and Utility of the Granulocyte-Macrophage Colony-Stimulating Factor Gene into Monocytes and Dendritic Cells by Adeno-Associated Virus. Journal of Interferon and Cytokine Research. 20:21–30.2000).

[0093] C. AAV-HEK293 cells containing adenovirus genes (E1, E2A, E4, VAI and VAII genes) integrated in the cell chromosome and expressed: established by Liu Yong, the inventor of this patent applic...

Embodiment 3

[0113] Embodiment 3, recombinant adeno-associated virus AAV / HPV-16 E7 mm Observation on tumorigenicity of primary cervical epithelial cells infected by virus and AAV / HPV-16 E7 virus

[0114] Materials and their sources:

[0115] A. rAAV virus: the recombinant adeno-associated virus (AAV / HPV-16 E7 that embodiment 2 obtains mm virus and AAV / HPV-16E7 virus).

[0116] B. Keratinocyte-SCF cell culture medium: purchased from American Life Technology Company.

[0117] C. Primary cervical epithelial cells: obtained by separating from normal cervical epithelial tissue by conventional methods.

[0118] Tumorigenicity Observation Experiment

[0119] Put the primary cervical epithelial cells into a 10.0cm cell culture dish, immediately add 10mL Keratinocyte-SCF cell culture medium, and culture them in a carbon dioxide incubator at 37°C. After the cells are completely attached to the wall, take out the culture dish, remove 7mL of the culture medium, and then add the recombinant adeno-as...

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Abstract

The invention discloses a recombinant adeno-associated viral vector with HPV-16 (human papillomavirus-16) E7<mm> antigen genes and a method for constructing the recombinant adeno-associated viral vector. The method includes mutating tumorigenicity HPV-16 E7 antigen genes at multiple points to obtain non-tumorigenicity E7 antigen genes; inserting the mutated genes into an adeno-associated viral vector without structural genes to obtain the recombinant adeno-associated viral vector. The recombinant adeno-associated viral vector and the method have the advantages that the mutant HPV-16 E7<mm> antigen genes carried by recombinant adeno-associated viruses can be delivered into monocyte-dendritic cell lines to be used for stimulating effect cells of immune systems; as proved by experiments, growth of HPV-16 E7 positive cells can be effectively inhibited by CTL (cytotoxic lymphocytes) induced by DC (dendritic cells) infected by the recombinant adeno-associated viruses for patients in in-vitro and in-vivo manners, or the HPV-16 E7 positive cells can be effectively killed by the CTL for the patients in the in-vitro and in-vivo manner, and tumorigenicity can be prevented; the recombinant adeno-associated viral vector or associated products can be used for preparing anti-tumor medicine for treating HPV-16 infection or diseases associated with the HPV-16 infection.

Description

technical field [0001] The present invention relates to a carrier in the biological field and its application, in particular to a human papillomavirus type 16 (Human Papillomavirus Type 16, HPV-16) multi-point mutant type E7 mm The recombinant adeno-associated virus vector (rAAV) of the antigen gene and its construction method and its application in the preparation of anti-HPV-16 infection and related diseases (such as tumors caused by HPV-16 infection) therapeutic drugs. , its construction method and its application in the preparation of medicines against HPV-16 infection and related diseases. Background technique [0002] The genetic structure of adeno-associated virus (AAV) has been identified. In 1983, Samulski et al. described the terminal repeat segment of AAV (upstream 5' segment, downstream 3' segment) (Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmamids: gene correction within the terminal repeats of AAV. C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N7/01C12N5/10A61K48/00A61K39/12A61P31/20A61P35/00
Inventor 刘勇陈巧林曾昭鹏高洪吉龚研浩董文娟张慧
Owner GUANGDONG TOPHEALTH BIOTECH
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