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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: 2017-04-26
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] Embodiment 1, the 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 the 41-289 nucleotides of sequence 2 in the sequence listing, and HTRP2 is encoded by the HTRP2 gene shown in the 47-3817 nucleotides of the 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 the HTRP1 gene, and the pHW2000 was digested with AarI to obtain the pHW2000 backbone vector 1, and the DNA fragment containing the HTRP1 gene was connected to the pHW2000 backbone vector 1 to obtain Recombinant vector, the recombinant vector is named pHW-HTRP...

Embodiment 2

[0114] Example 2, the specific effect of recombinant influenza virus rFLU-HTRP in liver cancer cells

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

[0116] Cells were seeded in a 96-well plate containing medium, the amount of medium in each well was the same, one cell per well, two replicate wells for each cell, and 10 wells per well 4 cells at 37°C, 5% CO 2 After culturing for 24 hours, each cell was inoculated with rFLU-HTRP suspension (each cell was inoculated with an equal volume of PBS as a control) according to the MOI of living cell TCID50 as 10...

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

The invention discloses a preparation method and application of a recombinant oncolytic influenza virus. The recombinant oncolytic influenza virus provided by the invention expresses HTRP1 and HTRP2; the HTRP1 is: (A1) protein shown by the sequence 1; (A2) protein derived from (A1) and obtained by substituting, deleting and / or adding one or more amino acid residues in the sequence 1; or (A3) fusion protein obtained by connecting the N terminal or / and C terminal of (A1) or (A2) with a label; and HTRP2 is: (B1) protein shown by the sequence 3; (B2) protein derived from (B1) and obtained by substituting, deleting and / or adding one or more amino acid residues in the sequence 3; or (B3) fusion protein obtained by connecting the N terminal or / and C terminal of (B1) or (B2) with a label. The recombinant oncolytic influenza virus provided by the invention can realize targeted killing of hepatoma cells without influencing normal host cells and can be applied to the targeted therapy of hepatoma.

Description

technical field [0001] The invention relates to the preparation method and application of 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 and limited effects. Cells also cause a lot of lethality and other issues. Therefore, the great challenge for tumor treatment is how to efficiently and specifically kill tumor cells without killing normal cells and tissues. With the development of molecular biology and people's new understanding of tumors at the gene level, new tumor treatment methods have emerged as the times require. In the 1990s, gene therapy began to be applied to the treatment of many diseases, and achieved certain curative effect in some genetic diseases....

Claims

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Application Information

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Patent Type & Authority Applications(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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