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Recombinant herpes simplex virus capable of excreting angiostatin and endostatin protein and its application in treating lung cancer

An endostatin, angiostatin technology, applied in application, gene therapy, virus/phage and other directions, can solve the problems of short expression time, lack of specificity, host infection, etc., achieve good genetic stability, inhibit virus growth, Infectious effect

Inactive Publication Date: 2009-05-20
罗益(无锡)生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But its shortcomings are also very obvious: 1) As mentioned above, after the retrovirus is packaged and introduced into cells, it is possible to restore the wild-type retrovirus, thereby producing a virus with replication ability, and the host has the risk of virus infection; 2) Since the retrovirus is randomly integrated into the host genome, it may cause "insertion mutagenesis"-it can trigger the activation of some proto-oncogenes near the insertion site and induce cancer; 3) The ability of retrovirus to package foreign DNA is very Limited, it generally can only package exogenous DNA less than 8kb; 4) Retroviruses also require the corresponding receptors of retroviruses on the surface of target cells
For adenovirus, its disadvantages are also very obvious: 1) adenovirus can infect almost all cells, so it lacks specificity; 2) because adenovirus cannot be integrated into the host chromosome, it cannot continuously express the desired product, express The time is short, and repeated administration of carrier preparations is required; 3) The virus particles of adenovirus can cause a strong immune response, which may stimulate the immune response, so that the effect of repeated treatment will gradually decrease
[0008] In addition, viral vectors commonly used in tumor therapy, such as retroviruses and adenoviruses, are non-replicative viral vectors, which limit the spread of viruses in tumors because they cannot achieve tumor-specific replication, and can only rely entirely on their The effect of the gene carried on the tumor, so its clinical effect is very limited

Method used

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  • Recombinant herpes simplex virus capable of excreting angiostatin and endostatin protein and its application in treating lung cancer
  • Recombinant herpes simplex virus capable of excreting angiostatin and endostatin protein and its application in treating lung cancer
  • Recombinant herpes simplex virus capable of excreting angiostatin and endostatin protein and its application in treating lung cancer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: Construction of recombinant virus AE618

[0063] (a) the preparation method of the F strain viral DNA of HSV-1 virus:

[0064] The F strain of HSV-1 virus used for recombination was purchased from ATCC Company of the United States. The virus was used to infect cultured Vero cells (ATCC, USA) under the condition of MOI=1. After 24-36 hours, when most of the cells showed cytopathic changes (CPE), the cells were collected and transferred to a 15ml centrifuge tube to remove the cell culture medium by centrifugation. Add 0.3ml of cell lysate (containing: 100Mm NaCl, 10Mm Tris.HCl pH 8, 25Mm EDTA pH8 and 0.5% SDS) to lyse the infected cells and treat them with RNaseA (10mg / ml) at 65°C for 1 hour. The phenol was then extracted with an equal volume of phenol / chloroform (1:1) mixture, the aqueous solution containing DNA was transferred to a new centrifuge tube, and 1 / 2 volume of 7.5M ammonium acetate and 2 times the volume of absolute alcohol were added. After...

Embodiment 2

[0089] Example 2: Detection of fusion protein expression in cancer cells infected with AE618

[0090] Before infection, the lung cancer A549 cells (1×10 6 )4 hours. Then they were infected with G207, AE618, or blank control (each infection titer M.O.I.=1.0), and the infected cells were cultured for 24 hours. Rinse the cultured cell samples twice with PBS, then gently scrape the cells from the plate, and use cell lysis buffer (50mM Tris-HCl pH7.5, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1 %SDS) to lyse the cells. Whole cell lysates were boiled and protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA). Take an equal amount of protein (100 μg) samples and perform electrophoresis separation on a 15% acrylamide gel under reducing conditions. The protein after electrophoresis was transferred to Immobilon-P transfer membrane (Millipore, Bedford, MA), and then the membrane was placed in 5% skimmed milk powder to block, and at 4°C, it was ...

Embodiment 3

[0092] Embodiment 3: Detection of the cell lysis effect of recombinant virus AE618

[0093] The A549 and H460 lung cancer cell lines (1×10 5 ) were placed in a 12-well tissue culture plate, cultured at 37°C for 4 hours, and then the cells were infected with various concentrations of G207 and AE618 recombinant viruses and a blank control, and the cells were continued to be cultured for several days. After trypsinization, cells were collected and resuspended in PBS solution. Add an equal volume of PBS containing 0.1% trypan blue to the cell suspension. Viable cell counts were then performed using a hemocytometer. All cell counts are the average of 3 sample counts.

[0094] The cell count results of H460 lung cancer cells from 1 to 5 days after infection showed that compared with the blank control, the number of living cells in the group treated with virus from the second day after infection decreased significantly (p0.05, Figure 2A ).

[0095] In addition, the H460 cell l...

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Abstract

The present invention relates to a recombinant herpes simplex virus and a method of gene treat of human tumour by using the recombinant virus, particularly relates to a recombinant herpes simplex virus with blood vessel chalone and endodermis chalone albumen, and its application in treating lung cancer. The invention also relates to medicine compound containing the recombinant virus. The recombinant herpes simplex virus of the invention can represent blood vessel chalone and endodermis chalone amalgamation albumen having effect of restraining the generation of blood vessel in the tumour cell, and the virus disintegratively increases in the tumour, such that the purpose of treating lung cancer by restraining the generation of blood vessel and disintegratively decomposing tumour cell is achieved.

Description

technical field [0001] The invention relates to a recombinant herpes simplex virus and a gene therapy method for human tumors using the recombinant virus. In particular, it relates to a recombinant herpes simplex virus carrying angiostatin and endostatin proteins, and its use in the treatment or prevention of lung cancer. It also relates to a pharmaceutical composition containing the recombinant virus. Background technique [0002] Lung cancer is one of the most common malignant tumors in humans, and its occurrence, development and metastasis are extremely complex processes of abnormal regulation of multiple genes. Most of the disease is not diagnosed in the early stage, so the treatment effect is often poor. The latest statistics show that the rate of increase in the mortality rate of lung cancer ranks first among all types of tumors, reaching 17.54 / 100,000 in the 1990s, which shows that it is increasingly harmful to human health. In recent years, with the development of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/869A61K48/00A61P35/00
Inventor 瞿伯荣贾韦国强东杨振达
Owner 罗益(无锡)生物制药有限公司
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