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Human papillomavirus (HPV) L1-based recombinant adenovirus for preventing and treating esophagus cancer

A technology of recombinant adenovirus and adenovirus, used in gene therapy, antiviral agents, genetic engineering, etc.

Inactive Publication Date: 2012-08-29
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no preventive and therapeutic vaccine research for esophageal cancer in the world. In most high-risk HPV-related cancers and precancerous lesions, L1 protein is continuously expressed in infected tumor cells, so it can be developed HPV L1 Vaccine for Esophageal Cancer

Method used

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  • Human papillomavirus (HPV) L1-based recombinant adenovirus for preventing and treating esophagus cancer
  • Human papillomavirus (HPV) L1-based recombinant adenovirus for preventing and treating esophagus cancer
  • Human papillomavirus (HPV) L1-based recombinant adenovirus for preventing and treating esophagus cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of codon-optimized HPV18L1 gene

[0045] 1.1 Analysis of the wild-type HPV18L1 gene shows that its codon usage preference is quite different from that of mammals, which will lead to inefficient use of isoform tRNA in host cells, thereby reducing the speed of protein translation. Our study also proved that the expression level of the wild-type HPV18L1 gene in mammalian cells is extremely low, even under the mediation of adenovirus vector, no obvious protein expression can be detected by Western blot. Therefore, optimizing the codons of the HPV18L1 gene can increase the expression level of the gene in mammalian cells by increasing the usage efficiency of the isoform tRNA and mutating the inhibitory elements in the gene sequence. Referring to the principle of human dominant codon usage, without changing the amino acid sequence, codon optimization was performed on the nucleotide sequence (1524bp) of the full-length coding region of the wild-type HPV18L...

Embodiment 2

[0046] Example 2 Construction of Replication Defective Recombinant Adenovirus Containing Codon-optimized HPV18L1 Gene

[0047] The construction process of the recombinant adenovirus containing the codon-optimized HPV18L1 gene replication defect is described in detail step by step (see figure 2 ).

[0048] 2.1 Construction of recombinant adenoviral shuttle plasmid containing codon-optimized HPV18L1 gene

[0049] (1) Preparation of Escherichia coli DH5α competent cells:

[0050] Pick a single colony of DH5α from a fresh plate cultured at 37°C for 16-20 hours, inoculate it in 5 ml of LB medium without antibiotics, and culture it overnight (12-16 hours) at 37°C with vigorous shaking. On the next day, draw 0.5ml from the above-mentioned culture and transfer it to 50ml LB medium at a ratio of 1:100 to continue culturing for about 3h. When the OD600 value of the bacterial solution is 3, transfer the bacteria to a sterile, Place in an ice-cooled 50ml centrifuge tube for 30 minutes....

Embodiment 3

[0063] Example 3 Identification and virus titer determination of recombinant adenovirus rAd-mod.HPV18L1

[0064] 3.1 Identification of recombinant adenovirus rAd-mod.HPV18L1

[0065] Use the PCR method to detect the insertion of the mod.HPV18L1 gene in the recombinant adenovirus: Take 50 μl of the virus supernatant and add 2 μl proteinase K (20 mg / ml), bathe in 55 ° C water for 1 hour, boil for 10 minutes, take 2 μl and dilute 10 times and 100 times, and then use Stock solution, 10-fold dilution, and 100-fold dilution were used as templates for PCR reactions. The upstream primer is 316F (sequence: 5'-agctagatctatggctctgtggcggcccagcgac-3'), the downstream primer is 316R (sequence: 5'-atttgtcgactcacttgcgggctctcacgcgcacg-3'), and the mod. 3). At the same time, take part of the virus infection according to 4 × 10 5 293 cells per hole were spread in a six-well plate. When the cells were completely damaged, the medium was centrifuged, and the adherent cells and exfoliated cells w...

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Abstract

The invention relates to human papillomavirus (HPV) L1-based recombinant adenovirus for preventing and treating esophagus cancer, which belongs to the field of biomedical technology. The invention provides an optimized genetic sequence of main capsid protein of code HPV18-type (HPV18) and replication-defective recombinant HPV18L1 adenovirus vaccine containing the genetic sequence, and provides a method for preparing the recombinant adenovirus. The recombinant HPV18L1 adenovirus and the recombinant HPV16L1 adenovirus vaccine (patent No. ZL200610056900.3) can be used for preventing and treating the esophagus cancer.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. The invention relates to a replication defective recombinant adenovirus containing codon-optimized HPV18L1 gene and a preparation method thereof. Background of the invention [0002] Esophageal cancer is one of the six most common malignant tumors in the world. Significant regional distribution differences (500-fold difference in incidence between high and low incidence areas) are its prominent epidemiological characteristics. Of the more than 400,000 newly diagnosed patients with esophageal cancer every year in the world, more than half occur in China. The Taihang Mountain area at the junction of Henan, Hebei and Shanxi provinces is an area with a very high incidence and mortality rate of esophageal cancer in the world. Research and development of preventive and therapeutic vaccines for esophageal cancer is an important key measure to reduce the incidence and mortality of esophageal cancer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/37C12N7/01C12N15/861A61K39/12A61K48/00A61P31/20A61P35/00
Inventor 曾毅周玉柏程江李泽琳郭艳涛李劲涛
Owner BEIJING UNIV OF TECH
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