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Carrying human papillomavirus type 16 mutant e7 m91 Recombinant adeno-associated virus vector of antigen gene and its construction method and application

A technology of human papillomavirus, hpv-16e7m91, which is applied in the direction of virus antigen components, virus/bacteriophage, application, etc., and can solve problems such as safety risks

Active Publication Date: 2017-06-13
GUANGDONG TOPHEALTH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the use of wild-type HPV-16 E7 antigen to stimulate the occurrence of immune responses in vivo and in vitro has certain risks in terms of safety.

Method used

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  • Carrying human papillomavirus type 16 mutant e7 <sub>m91</sub> Recombinant adeno-associated virus vector of antigen gene and its construction method and application
  • Carrying human papillomavirus type 16 mutant e7 <sub>m91</sub> Recombinant adeno-associated virus vector of antigen gene and its construction method and application
  • Carrying human papillomavirus type 16 mutant e7 <sub>m91</sub> Recombinant adeno-associated virus vector of antigen gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1, Construction of recombinant adeno-associated virus vectors AAV / HPV-16 E7 and AAV / HPV-16 E7 m91

[0062] 1. Materials:

[0063] 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 (CTGCGCTGG, the purpose...

Embodiment 2

[0073] Embodiment 2, preparation of recombinant adeno-associated virus (rAAV) and virus titer determination

[0074] Materials and their sources:

[0075] A. Carrying HPV-16 E7 constructed in Example 1 m91 and HPV-16 E7 antigen gene recombinant adeno-associated virus vector (AAV / HPV-16 E7 m91 and AAV / HPV-16 E7).

[0076] B. Auxiliary plasmid pHelper containing the Rep gene and Cap gene of AAV: successfully constructed by the inventors of this patent application (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).

[0077] C. AAV-HEK293 cells containing adenovirus genes (E1, E2A, E4, VAI and VAII genes) integrated in the cell chromosome and expressed: established by the inventors of this patent application (Liu, Y., Santin AD...

Embodiment 3

[0097] Embodiment 3, recombinant adeno-associated virus rAAV (AAV / HPV-16 E7 m91 Observation on tumorigenicity of primary cervical epithelial cells infected by virus and AAV / HPV-16 E7 virus

[0098] Materials and their sources:

[0099] A. rAAV virus: AAV / HPV-16 E7 obtained in Example 2 m91 virus and AAV / HPV-16 E7 virus.

[0100] B. Keratinocyte-SCF cell culture medium: purchased from American Life Technology Company.

[0101] C. Primary cervical epithelial cells: isolated from normal cervical epithelial tissue by conventional methods.

[0102] Tumorigenicity Observation Experiment

[0103] 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 were completely adhered to the wall, take out the culture dish, remove 7mL of the culture medium, and add the recombinant adeno-associated virus AAV / HPV-16 E7 at a dose of 100MOI. m9...

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Abstract

The invention relates to a recombinant adeno-associated virus vector carrying a human papillomavirus type 16 (HPV-16) E7m91 antigenic gene and the construction method thereof. The recombinant adeno-associated virus vector is obtained after an E7 antigenic gene containing oncogenic HPV-16 is mutated into an oncogenicity-free E7 antigenic gene, and then the mutated gene is inserted into an adeno-associated virus vector with a structural gene removed. The recombinant adeno-associated virus can convey the carried mutant HPV-16 E7m91 antigenic gene into a monocyte-dendritic cell line to be used for stimulating effector cells of an immune system. Experiments prove that CTL induced by DC infected with the recombinant adeno-associated virus can effectively restrain the growth of HPV-16 E7 positive cells or kill the HPV-16 E7 positive cells in vitro and in vivo. The recombinant adeno-associated virus vector or relevant products can be used for preparing antineoplastic drugs for treating HPV-16 infection and relevant diseases.

Description

technical field [0001] The 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) mutant E7 m91 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 its related disease treatment drugs. 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 a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N15/37C12N7/01C12N5/10A61K39/12A61K48/00A61P31/20A61P35/00
Inventor 刘勇陈巧林曾昭鹏董文娟高洪吉龚研浩张慧卢敬
Owner GUANGDONG TOPHEALTH BIOTECH