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IL12 bicistronic virus construct for treating liver cancer and application and construction method of IL12 bicistronic virus construct

A technology for treating liver cancer and constructs, which is applied in the fields of biotechnology and gene therapy, and can solve the problems of not considering the effect of enhancing virus replication on oncolysis and the necessity of not considering it.

Inactive Publication Date: 2020-10-30
药鼎(北京)国际细胞医学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the present invention does not consider the necessity of the existence of IL-12 double subunits and cistrons, nor does it consider the promotion effect of virus replication on oncolysis, etc.

Method used

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  • IL12 bicistronic virus construct for treating liver cancer and application and construction method of IL12 bicistronic virus construct
  • IL12 bicistronic virus construct for treating liver cancer and application and construction method of IL12 bicistronic virus construct
  • IL12 bicistronic virus construct for treating liver cancer and application and construction method of IL12 bicistronic virus construct

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 - Construction of viral constructs

[0079] Restriction enzymes HindⅢ / SalⅠ / EcoRI / XbaⅠ in the present invention were purchased from New England Biolabs; PCR amplification reagents and cDNA synthesis kits were purchased from TARAKA Biotechnology (Beijing) Co., Ltd.; endotoxin-free plasmid extraction Kits DP117 and DP118 were purchased from TIANGN Biochemical Technology (Beijing) Co., Ltd.; pBHGlox_E1, 3Cre recombinant adenovirus backbone vector and pDC316 adenovirus vector were purchased from Changsha Youbao Biotechnology; IRES, IL12-P40, IL12-P35 , RGD-4C-IL12-P40-IRES-IL12-P35 and AFP fragments and all amplification primers were synthesized in Sangon Biotech (Shanghai); DNA recovery kits were purchased from Promega; NK cells and PSMPUW-IRES-GFP were purchased from the company Laboratory preservation and preparation.

[0080] The invention discloses a method for constructing a novel oncolytic virus for individualized targeted therapy of liver cancer, which com...

Embodiment 2

[0104] Example 2—oncolytic effect verification experiment

[0105] In the present invention, novel oncolytic virus Adv-Ade AFP+RGD-4C+p40-IRES-p35 It has a selective inhibitory effect on liver cancer cells, does not infect normal liver cells, and has a very low infection rate in other tumor cells.

[0106] 1.MTT experiment

[0107] MTT is a good method to reflect the viability of cells. The present invention uses MTT to detect (0,0.001,0.01,0.1,1Adv-Ade AFP+RGD-4C+p40-IRES-p35 The effect on the viability of liver cancer cell HepG2 and normal cell HL7702, found that this new virus Adv-Ade AFP+RGD-4C+p40-IRES-p35 Can significantly inhibit the viability of liver cancer cells HepG2, but has no effect on the viability of normal liver cells HL7702 (see figure 2 )

[0108] 2. Plate colony formation experiment

[0109] Clonogenicity is a unique property of malignant cells. Plate colony formation assay can well reflect the colony formation ability of tumor cells. The present i...

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Abstract

The invention relates to a virus construct for treating liver cancer and an application and construction method of the virus construct. The invention also relates to a novel oncolytic virus for individualized targeted treatment on liver cancer and a construction method thereof. The construct of the present invention comprises an AFP promoter, an RGD-4C gene, an IL-12-p40 gene, an IRES gene, an IL-12-p35 gene, and optionally a glutamyl transpeptidase gene and a tumor-associated antigen gene in an effective ligation manner.

Description

Technical field: [0001] The present invention relates to the field of biotechnology and gene therapy, specifically, the present invention relates to a viral construct containing IL12 bicistronic for treating liver cancer and its application and construction method. The invention also relates to a novel oncolytic virus for individualized targeted therapy of liver cancer and its construction method. Background technique: [0002] Liver cancer refers to malignant tumors of the liver and is generally divided into two categories: primary liver cancer and secondary liver cancer. Primary liver cancer originates from the epithelial or mesenchymal tissue of the liver, and is a high-incidence cancer in my country. Compared with primary liver cancer, secondary or metastatic liver cancer mainly refers to cancers that have metastasized to various organs of the body and invade the liver. There are many reasons for inducing liver cancer, such as hepatitis B and C virus infection, drinking...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/66C12N15/24A61K48/00A61K38/20A61P35/00A61P1/16
CPCA61K38/20A61K48/0058A61P1/16A61P35/00C07K14/54C12N15/66C12N15/86C12N2710/10343C12N2800/107C12N2840/203
Inventor 宫晓艳周海涛秦苗苗刘进稳
Owner 药鼎(北京)国际细胞医学技术有限公司
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