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Construction and preparation of a recombinant adeno-associated virus vector and its application value

A technology of viral vectors and human papillomaviruses, applied in the direction of viruses/bacteriophages, viruses, applications, etc., can solve problems such as safety risks, and achieve the effect of broad application prospects

Active Publication Date: 2021-10-22
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 the occurrence of immune responses with wild-type HPV-16E6 antigen in vivo and in vitro

Method used

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  • Construction and preparation of a recombinant adeno-associated virus vector and its application value
  • Construction and preparation of a recombinant adeno-associated virus vector and its application value
  • Construction and preparation of a recombinant adeno-associated virus vector and its application value

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction of recombinant adeno-associated virus vector AAV / HPV-16E6 m

[0050] 1. Materials and their sources:

[0051] A. Adeno-associated virus vector: It has the macrophage virus (CMV) promoter and the complete repeat terminal fragment (TR) sequence at both ends of AAV type 2, and is inserted at the 75th nucleotide sequence of both ends of the TR 9 nucleotide fragments (CTGCGCTGG, the purpose is to improve the stability of the recombinant AAV virus (rAAV) and the replication efficiency of the virus). Since there are no AAV structural genes (Rep and Cap), the AAV vector has lost the function of integrating into the chromosome. The AV vector was successfully constructed by the inventor of this patent application (see Chinese patent ZL201110125683.X for the construction method).

[0052] B. Human HPV-16E6 antigen-positive tissue: The cervical cancer tissue used in the experiment was surgically resected, and immunohistochemistry confirmed that it was positi...

Embodiment 2

[0066] Example 2 Preparation of recombinant adeno-associated virus AAV / HPV-16E6m and virus titer determination Materials and sources thereof:

[0067] A. The recombinant adeno-associated virus vector (AAV / HPV-16E6m) carrying the HPV-16E6m gene constructed in Example 1.

[0068] 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).

[0069] 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 application, et al. (Liu, Y., Santin AD ., Mane M....

Embodiment 3

[0088] Example 3 Observation on tumorigenicity of primary cervical epithelial cells infected by recombinant adeno-associated virus AAV / HPV-16E6 virus and AAV / HPV-16E6m virus

[0089] Materials and their sources:

[0090] A. rAAV virus: the recombinant adeno-associated virus AAV / HPV-16E6m virus obtained in Example 2. Similarly, AAV / HPV-16E6 virus can be obtained

[0091] B. Keratinocyte-SCF cell culture medium: purchased from American Life Technology Company.

[0092] C. Primary cervical epithelial cells: isolated from tissues that have been proven to be free of HPV.

[0093] Tumorigenicity Observation Experiment

[0094] Primary cervical epithelial cells were cultured in a 10.0 cm cell culture dish containing 10 mL of Keratinocyte-SCF cell culture medium 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, add the recombinant adeno-associated virus AAV / HPV-16E6m virus or AAV...

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Abstract

The invention belongs to the field of molecular biology, and specifically relates to the construction and preparation of a recombinant adeno-associated virus vector and its application value. The invention is obtained by mutating the tumorigenic HPV-16 E6 antigen gene into a non-tumorigenic E6 antigen gene, and then inserting the mutant gene into the adeno-associated virus vector whose structural gene has been deleted. The recombinant adeno-associated virus can be used to deliver the mutant HPV-16 E6 antigen gene carried by it into the monocyte-dendritic cell line, which is used to stimulate the effector cells of the immune system. Experiments have proved that the cytotoxic T lymphocytes induced by DCs infected by the recombinant adeno-associated virus can effectively kill HPV‑16 E6 positive cells and tumor cells without tumorigenicity. The recombinant adeno-associated virus vector and related products of the present invention can also be used for anti-HPV-16 infection and related targeted cellular immunotherapy.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to the construction and preparation of a recombinant adeno-associated virus vector carrying a mutant E6 antigen gene of human papillomavirus type 16 and its application value. 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. Cell. 33:135-143.). In 1984, Hermonat et al. described the low infectious particle (Lip) gene and envelope (Cap) gene of AAV (Hermonat PL, LabowMA, Wright R, Berns KI, Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.JVirol.51:329-339.Her...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N15/37C12N7/01C12N5/10A61K39/12A61P31/20A61P35/00C12R1/91
CPCA61K39/12A61P31/20A61P35/00C07K14/005C12N7/00C12N15/86C12N2710/20022C12N2710/20034C12N2750/14121C12N2750/14143
Inventor 刘勇陈汉森钟海迪郭庐庆
Owner GUANGDONG TOPHEALTH BIOTECH
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