Recombinant herpes simplex virus, method for producing the same, and its applications

Abstract

This invention relates to the field of genetic engineering. Recombinant herpes simplex virus and method for preparing the same. The recombinant herpes simplex virus and its uses are disclosed. The recombinant herpes simplex virus is a vector and a DPD gene. The vector includes a nucleotide sequence for reducing the expression level of the offspring. It is a herpes simplex virus that contains the CD gene but lacks the gene encoding ICP47. The insertion site of the nucleotide sequence for reducing the expression level of the DPD gene is the aforementioned vector This is the deletion site of the gene encoding ICP47 in the recombinant herpes simplex virus. Russ introduced shRNA that interferes with DPD into HSV-CD, thereby enabling the development of CD / 5-FC glioma cells. It enhances the killing ability against the target while simultaneously interfering with the metabolism of glioma cells.

Claims

1. It is a recombinant herpes simplex virus, The recombinant herpes simplex virus reduces the expression levels of the vector and the DPD gene. It contains the nucleotide sequence, The vector is a herpes simplex virus that lacks the gene encoding ICP47 and contains the CD gene. It is a virus, and the insertion of a nucleotide sequence to reduce the expression level of the DPD gene. The site is the deletion site of the gene encoding ICP47 in the aforementioned vector, The nucleotide sequence for reducing the expression level of the DPD gene is sequence number 1, sequence number Recombinant herpes simplex virus characterized by being represented by code 2, SEQ ID NO: 3, or SEQ ID NO:

4. 。

2. A recombinant herpes simplex virus according to claim 1, The insertion site of the CD gene in the aforementioned vector is at the deletion site of the gene encoding ICP34.

5. A recombinant herpes simplex virus with certain characteristics.

3. A recombinant herpes simplex virus according to claim 1, The DPD gene is characterized by having the nucleotide sequence shown in Sequence ID No.

5. Herpes simplex virus.

4. A recombinant herpes simplex virus according to claim 1, The aforementioned CD gene is characterized by having the nucleotide sequence shown in Sequence ID No.

6. Herpes simplex virus.

5. A recombinant herpes simplex virus according to claim 1, The aforementioned vector is characterized by being obtained by the following method: recombinant herpes simplex virus Rus: Knock out the gene encoding ICP34.5 in herpes simplex virus, and ICP34.5 The CD gene is inserted at the knockout position of the gene encoding the gene, and a simple gene containing the CD gene is created. The process of obtaining the Rupes virus, The gene encoding ICP47 in the herpes simplex virus, including the aforementioned CD gene, is knocked out. The process of outputting and obtaining the vector.

6. A method for producing recombinant herpes simplex virus according to any one of claims 1 to 5, Knockout of the gene encoding ICP47 in herpes simplex virus, including the CD gene. The process of obtaining a vector, A nucleotide sequence for reducing the expression level of the DPD gene is inserted into the aforementioned vector. Including the process, The nucleotide sequence insertion site for reducing the expression level of the DPD gene is the This is the site where the gene encoding ICP47 is knocked out in the kutar. The nucleotide sequence for reducing the expression level of the DPD gene is sequence number 1, sequence number Indicated by code 2, sequence number 3, or sequence number 4, A manufacturing method characterized by the following features.

7. A method for producing recombinant herpes simplex virus according to claim 6, The herpes simplex virus containing the CD gene has ICP34.5 in the herpes simplex virus. The gene encoding the aforementioned ICP34.5 is knocked out. The CD gene is inserted at the specified position to obtain a herpes simplex virus containing the CD gene. Characteristic manufacturing method.

8. A method for producing recombinant herpes simplex virus according to claim 7, The CD gene was synthesized, and the CD gene was converted to pSP72△ICP34.5CMVWPRE vector using Hind III and Xho I. The process involves cloning to obtain pSP72△ICP34.5CMVCDWPRE, Co-transfection of BHK cells with pSP72△ICP34.5CMVCDWPRE and HSV-1△ICP34.5CMVEGFP was performed. Furthermore, viral plaques that do not express green fluorescence have the EGFP gene in the protovirus and the CD gene This indicates that substitution occurred by homologous recombination. The HSV-1△ICP34.5CMVCDWPRE viral vector was purified and amplified, and the full-length DNA of the vector was extracted. The process involves obtaining a herpes simplex virus containing the CD gene by extracting it, A manufacturing method characterized by further comprising the following.

9. Recombinant herpes simplex virus according to any one of claims 1 to 5, or any of claims 6 to 8 Recombinant herpes simplex virus produced by the method described in item 1, for the treatment of cerebral glioma Applications in the manufacture of therapeutic drugs.

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