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Preparation method and application of recombinant oncolytic influenza virus

A kind of influenza virus, virus technology, applied in the biological field, can solve problems such as vigorous growth

Active Publication Date: 2019-08-23
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, many tumor cells are resistant to Ras-activated apoptosis, but retroviruses thrive in such cells due to the inhibition of double-stranded RNA-activated protein kinase R (PKR)

Method used

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  • Preparation method and application of recombinant oncolytic influenza virus
  • Preparation method and application of recombinant oncolytic influenza virus
  • Preparation method and application of recombinant oncolytic influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1. Rescue of recombinant influenza virus

[0101] The present invention provides a recombinant influenza virus (recombinant oncolytic influenza virus), which expresses proteins named HTRP1 and HTRP2 respectively; HTRP1 is a protein whose amino acid sequence is sequence 1, and HTRP2 is a protein whose amino acid sequence is sequence 3. HTRP1 is encoded by the HTRP1 gene shown in nucleotides 41-289 of sequence 2 in the sequence listing, and HTRP2 is encoded by the HTRP2 gene shown in nucleotides 47-3817 of sequence 4 in the sequence listing.

[0102] 1. Construction of recombinant vector

[0103] The DNA molecule shown in sequence 2 was digested with AarI to obtain a DNA fragment containing HTRP1 gene, pHW2000 was digested with AarI to obtain pHW2000 backbone vector 1, and the DNA fragment containing HTRP1 gene was ligated with pHW2000 backbone vector 1 to obtain The recombinant vector is named pHW-HTRP1, and pHW-HTRP1 expresses HTRP1 shown in sequence 1.

[0104] The DN...

Embodiment 2

[0114] Example 2. Specific effect of recombinant influenza virus rFLU-HTRP in liver cancer cells

[0115] The rFLU-HTRP in step 2 of Example 1 was resuspended in PBS to obtain an rFLU-HTRP suspension. MTS kit (Promega) was used to detect the cytotoxicity of recombinant influenza virus rFLU-HTRP on liver cancer cells at different multiplicity of infections. The liver cancer cells used were SMMC-7721, HepG2, MHCC-97L and HuH-7.5, and human normal liver cells were used. L02 served as a control. The experiment was repeated three times, and the specific steps for each repeated experiment are as follows:

[0116] Inoculate the cells in a 96-well plate containing medium. The amount of medium in each well is the same, one type of cell per well, two replicate wells of each type of cell, 10 per well 4 Cells at 37℃, 5% CO 2 After incubating for 24 hours, each cell was inoculated with rFLU-HTRP suspension (an equal volume of PBS was inoculated as a control) according to the multiplicity of in...

Embodiment 3

[0129] Example 3. Anti-tumor effect of recombinant influenza virus rFLU-HTRP in nude mice bearing liver cancer transplantation

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Abstract

Provided are a preparation method and application of a recombinant oncolytic influenza virus expressing HTRP1 and HTRP2, wherein the HTRP1 is: (A1) a protein represented by SEQ ID No.1; (A2) a protein derived from (A1) by substitution, deletion and / or addtion of one or more amino acid residues in SEQ ID No.1; or (A3) a fusion protein obtained by connecting the N terminal or / and C terminal of (A1) or (A2) with a label; the HTRP2 is: (B1) a protein represented by SEQ ID No.3; (B2) a protein derived from (B1) by substitution, deletion and / or addtion of one or more amino acid residues in SEQ ID No.3; or (B3) a fusion protein obtained by connecting the N terminal or / and C terminal of (B1) or (B2) with a label. The recombinant oncolytic influenza virus can be used in targeting killing of hepatoma cells without influencing normal host cells, and thus can be used in hepatoma targeting therapy.

Description

Technical field [0001] The invention relates to a preparation method and application of a recombinant oncolytic influenza virus in the field of biotechnology. Background technique [0002] Tumor is a "killer" that seriously endangers human health. The existing clinical treatment methods include surgery, radiotherapy, chemotherapy, targeted drugs and other methods. Although they have a certain degree of clinical efficacy, they have large side effects, limited effects, and normal human health. Cells also cause problems such as great lethality. Therefore, the huge challenge facing tumor treatment is how to efficiently and specifically kill tumor cells, but not normal cell tissues. With the development of molecular biology and people's new understanding of tumors at the genetic level, new tumor treatment methods have emerged. In the 1990s, gene therapy began to be used in the treatment of a variety of diseases, and achieved certain effects in some genetic diseases. These advances ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/63C12N15/44C12N15/62A61K35/768A61P35/00
Inventor 杨鹏辉王希良张绍庚任天宇张培瑞王兆海孙芳
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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