Construction and application of oncolytic influenza virus targeting liver cancer
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A technology of influenza viruses and strains, applied in the direction of viruses, applications, viral peptides, etc., can solve problems such as ineffective treatment and complicated tumor mechanisms
Active Publication Date: 2019-07-26
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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However, the tumor mechanism is complex and the gene mutations are diverse. The introduction of one or two tumor suppressor genes is not enough to inhibit the growth of tumor cells, and the therapeutic effect is not obvious.
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Embodiment 1
[0097] Embodiment 1, the rescue of recombinant influenza virus
[0098] The invention provides a recombinant influenza virus, which expresses proteins whose names are respectively HRP1 and HRP2; HRP1 is a protein whose amino acid sequence is sequence 1, and HRP2 is a protein whose amino acid sequence is sequence 3, and HRP1 is derived from the sequence in the sequence table The HRP1 gene coded by the 51-623 nucleotides of 2, and the HRP2 coded by the HRP2 gene shown by the 35-2332 nucleotides of the sequence 4 in the sequence listing.
[0099] 1. Construction of recombinant vector
[0100] The DNA molecule shown in Sequence 2 was digested with AarI to obtain a DNA fragment containing the HRP1 gene, and the pHW2000 was digested with AarI to obtain the pHW2000 backbone vector 1, and the DNA fragment containing the HRP1 gene was connected to the pHW2000 backbone vector 1 to obtain Recombinant vector, the recombinant vector is named as pHW-HRP1, HRP1 shown in pHW-HRP1 expression ...
Embodiment 2
[0108] Example 2, the specific effect of recombinant influenza virus rFLU-HRP in liver cancer cells
[0109] 1. MTS method to detect the effect of recombinant influenza virus rFLU-HRP on liver cancer cells
[0110] The rFLU-HRP in step 2 of Example 1 was resuspended with PBS to obtain rFLU-HRP suspension. MTS kit (Promega) was used to detect the cytotoxic specificity of recombinant influenza virus rFLU-HRP to liver cancer cells at different multiplicity of infection. The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:
[0111] Inoculate HepG2, SMMC-7721 and L02 in a 96-well plate 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 for each type of cell, 10 wells per well 4 cells at 37°C, 5% CO 2 After culturing for 24 hours, each cell was inoculated with rFLU-HRP suspension (each cell was inoculated with an equal volume of PBS as a control)...
Embodiment 3
[0120] Example 3. Anti-tumor effect of recombinant influenza virus rFLU-HRP in nude mice bearing liver cancer
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Abstract
The invention discloses construction and application of liver cancer targeted oncolytic influenza viruses. The reassorted influenza viruses provided by the invention express proteins HRP1 and HRP2, wherein the protein HRP1 is A1) protein as shown in a sequence 1, A2) protein derived by the protein A1) and obtained by substitution, deletion and / or addition of one or more amino acid residues in the sequence 1, or A3) fusion protein obtained by connecting a tag to the N end or / and the C end of the protein A1) or A2); and the protein HRP2 is B1) protein as shown in a sequence 3, B2) protein derived by the protein B1) and obtained by substitution, deletion and / or addition of one or more amino acid residues in the sequence 3, or B3) fusion protein obtained by connecting a tag to the N end or / and the C end of the protein B1) or B2). The oncolytic reassorted influenza viruses provided by the invention can kill liver cancer cells in a targeted manner, do not have remarkable influence on normal host cells, and can be used for liver cancer targeted treatment.
Description
technical field [0001] The invention relates to the construction and application of an oncolytic influenza virus targeting liver cancer in the field of tumor gene therapy and virus therapy. Background technique [0002] Primary liver cancer is one of the most common malignant tumors in my country. The incidence of liver cancer in my country ranks first in the world, and nearly half of the world's incidence population is located in China. The existing treatment methods for liver cancer mainly include surgical resection, liver transplantation, local ablation therapy, transhepatic arterial chemoembolization, radiotherapy, molecular targeted therapy, comprehensive therapy, etc. Difficult to cure. Therefore, a major problem in the treatment of liver cancer is how to efficiently and specifically kill tumor cells while reducing damage to normal cells and tissues. With the development of molecular biology and people's new understanding of tumors at the gene level, new tumor treat...
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