Recombinant oncolytic gene-adenovirus targeting cancer and construction method and application of recombinant oncolytic gene-adenovirus

An adenovirus and gene technology, applied in the field of recombinant oncolytic gene-adenovirus targeting cancer and its construction, can solve the problems of high harm to patients, inability to select tumor cells, and high side effects, and achieve good therapeutic effects and anti-cancer effects. Good effect, low toxicity effect

Pending Publication Date: 2019-12-31
SHANGHAI YUANSONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current various gene carriers cannot specifically select tumor cells in the human body, nor can they efficiently express anti-cancer genes in tumor cells, resulting in weak gene therapy effects and the possibility of accidentally injuring normal human cells
[0004] In addition, for early cancers, the existing method is to use surgical methods to remove tumors, and the effect is ideal. However, some tumors have a long hidden period and are not easy to be detected early, and the best time for treatment is missed.
However, the method of surgery in the middle and late stages is no longer feasible, and chemotherapy or radiotherapy is generally used, while the traditional methods of radiotherapy and chemotherapy have the disadvantages of poor curative effect and high side effects
The current level of medical treatment, whether it is surgical treatment or drug treatment, is very harmful to the patient. In addition to relieving the patient's symptoms to a certain extent, it cannot effectively prolong the patient's survival time.

Method used

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  • Recombinant oncolytic gene-adenovirus targeting cancer and construction method and application of recombinant oncolytic gene-adenovirus
  • Recombinant oncolytic gene-adenovirus targeting cancer and construction method and application of recombinant oncolytic gene-adenovirus
  • Recombinant oncolytic gene-adenovirus targeting cancer and construction method and application of recombinant oncolytic gene-adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The construction method of recombinant oncolytic gene-adenovirus Ad-sp-E1A-ΔE1B-VGLL4 targeting cancer is as follows: Step 1, construction of plasmid pCA13-VGLL4

[0064] (1) PCR method to obtain the VGLL4 gene fragment

[0065] Using the plasmid pcDNA-flag-VGLL4 as a template, the VGLL4 gene fragment was obtained by PCR.

[0066] The PCR reaction system is:

[0067]

[0068] The reaction procedure of PCR is:

[0069]

[0070] PCR products were run on 1% agarose gel electrophoresis. PCR results such as figure 1 shown. Among them, lane 1 is DNA Marker (Trans2K Plus DNA Marker); lane 2 is H 2 O (negative control); Lane 3 is the PCR product of pcDNA-flag-VGLL4.

[0071] The results showed that the target band was between 909bp and 750bp to 1000bp, indicating that the VGLL4 gene fragment was successfully amplified.

[0072] (2) Purification of PCR products

[0073] Purify the PCR product of the VGLL4 gene fragment obtained by PCR in Step 1 (1) with an agarose ...

Embodiment 2

[0142] Example 2 Identification of Recombinant Oncolytic Gene-Adenovirus Ad-sp-E1A-ΔE1B-VGLL4

[0143] (1) Extract viral genomic DNA according to the instructions of the blood genome extraction kit

[0144] (2) PCR identification of the VGLL4 gene carried by Ad-sp-E1A-ΔE1B-VGLL4 in step 4

[0145] The PCR reaction system is as follows:

[0146]

[0147] The reaction procedure of PCR is:

[0148]

[0149] Run electrophoresis after PCR. The result is as Figure 9 shown. Among them, lane 1 is DNA Marker (Trans2K Plus DNAMarker); lane 2 is the PCR product of Ad-sp-E1A-ΔE1B-VGLL4.

[0150] The results showed that the target band was 891bp, slightly below 1000bp, which proved that VGLL4 was amplified successfully.

[0151] (3) PCR to identify whether there is wild virus of E1A wild-type promoter recombinant type in the recombinant oncolytic gene-adenovirus Ad-sp-E1A-ΔE1B-VGLL4 obtained in Step 4 construction.

[0152] The PCR reaction system is as follows:

[0153] ...

Embodiment 3

[0163] Example 3 Titer determination of recombinant oncolytic gene-adenovirus Ad-sp-E1A-ΔE1B-VGLL4

[0164] The principle of virus titer determination is to determine the number of virus with infectious activity by counting the number of hexon-positive cells by immunocytochemical method, and measure the titer of the adenovirus after small amplification and purification according to the instructions on the adenovirus titer kit. .

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Abstract

The invention discloses a recombinant oncolytic gene-adenovirus targeting cancer, and the recombinant oncolytic gene-adenovirus is Ad-sp-E1A-delta E1B-VGLL4. The recombinant oncolytic gene-adenovirusAd-sp-E1A-delta E1B-VGLL4 is driven by a survivin promoter to drive a virus E1A gene, an E1B gene region is knocked out off the virus genome at the same time, and a VGLL4 expression frame fragment isinserted on the basis. The invention also discloses a construction method and application in preparing drugs for treating cancer of the recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4.The recombinant oncolytic gene-adenovirus Ad-sp-E1A-delta E1B-VGLL4 of the invention specifically replicates in cancer cells with the aid of an adenovirus vector, and achieves good therapeutic effects in anticancer tests.

Description

technical field [0001] The present invention relates to the technical field of recombinant oncolytic gene-adenovirus, in particular to a cancer-targeting recombinant oncolytic gene-adenovirus and its construction method and application. Background technique [0002] Cancer seriously affects human health and development. Affected by medical and environmental conditions, cancer mortality in China is higher than the global average. Traditional tumor therapy has disadvantages such as poor curative effect, high mortality rate and high recurrence rate, so people began to place high hopes on new tumor treatment methods. Emerging tumor treatment strategies are also under constant development, and currently more recognized strategies include immunotherapy, gene therapy, and oncolytic virus therapy. [0003] Currently, only gene therapy or viruses that specifically target tumors are used to treat cancer, and their effects are very limited. However, inserting anti-cancer genes into t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861C12N15/66A61K35/761A61K38/17A61K48/00A61P35/00
CPCC12N7/00C12N15/86C12N15/66C07K14/4702A61K35/761A61K48/005A61K48/0008A61K38/1709A61P35/00C12N2710/10021C12N2710/10043C12N2710/10032A61K2300/00
Inventor 章康健方先龙刘新垣顾锦法倪爱明
Owner SHANGHAI YUANSONG BIOTECHNOLOGY CO LTD
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