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Recombinant II type herpes simplex virus vector, preparation method of recombinant II type herpes simplex virus vector, recombinant virus, medicinal composition and application

A technology of herpes simplex virus and carrier, applied in the application field of gene medicine, which can solve the problems of short production cycle, high oncolytic activity, and insufficient oncolytic activity

Active Publication Date: 2011-08-10
WUHAN BINHUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the shortcomings of the existing herpes simplex virus vectors prepared from wild-type herpes simplex virus type I, long production cycle and insufficient oncolytic activity, and provide a recombinant type II vector with short production cycle and high oncolytic activity Herpes simplex virus vector

Method used

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  • Recombinant II type herpes simplex virus vector, preparation method of recombinant II type herpes simplex virus vector, recombinant virus, medicinal composition and application
  • Recombinant II type herpes simplex virus vector, preparation method of recombinant II type herpes simplex virus vector, recombinant virus, medicinal composition and application
  • Recombinant II type herpes simplex virus vector, preparation method of recombinant II type herpes simplex virus vector, recombinant virus, medicinal composition and application

Examples

Experimental program
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preparation example Construction

[0085] The present invention also provides a method for preparing a recombinant type II herpes simplex virus vector, wherein the method comprises the following steps:

[0086] (1) Delete the ICP47 gene from the wild type II herpes simplex virus HG52 strain to construct HG52dICP47 recombinant type II herpes simplex virus:

[0087] a. extracting the full-length viral DNA of type II herpes simplex virus HG52 strain;

[0088] b. Construct the plasmid pdICP47H2 containing the upstream flanking region sequence and the downstream flanking region sequence of the ICP47 gene:

[0089] b1. Using the primers shown in Table 1, using the full-length viral DNA obtained in step a as a template, PCR amplifies the upstream flanking region sequence and the downstream flanking region sequence of the ICP47 gene;

[0090] Table 1

[0091]

[0092] b2. Insert the upstream flanking region sequence of the PCR product amplified in step b1 into the SmaI site of the pSP73 plasmid to obtain the pSP73IC...

Embodiment 1

[0203] Delete the ICP47 gene from the wild-type HSV-2 (HG52) genome to construct HG52dICP47 .

[0204] (1) DNA of purified HG52 wild-type virus

[0205] HG52 virus was cultured with BHK cells and DNAzol TM Genomic DNA Isolation Kit (Helena Biosciences Cat. No. DN127200) was used to purify viral DNA. BHK cells were grown in a 175 cm square culture flask, and the culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin. The culture conditions were 37°C, 5% carbon dioxide. When the cells were grown to 90% saturation, the virus was inoculated. Continue to incubate for 24-48 hours. When more than 90% of the cells have cytopathic changes, remove the culture medium and add 10ml of DNAzol. Use a 10ml pipette to suck and blow 5 times, and transfer the cell lysate to a 50ml Falcon test tube, add 5ml of 100% ethanol, and shake the test tube gently in a circular motion to fully analyze the viral DNA. Pick the DNA into another test tube with a tip...

Embodiment 2

[0271] This example illustrates the pharmaceutical composition of the invention.

[0272] The preparation of the injection will prepare a group of phosphate buffer solution according to the dosage shown in Table 3, and sterilize at 121°C for 20 minutes. Under sterile conditions, filter the herpes simplex virus stock solution of known titer in Example 1 with a microporous membrane of 0.45 microns to remove cell debris, collect the filtrate into a sterile centrifuge tube, and centrifuge at 8000 rpm for 1 hour, discard Remove the supernatant, and according to the virus titers shown in Table 3, disperse the obtained virus precipitate in the above-mentioned high-temperature sterilized phosphate buffer solution to obtain the injection solution of the present invention.

[0273] table 3

[0274] drug

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Abstract

The invention provides a recombinant II type herpes simplex virus vector. An ICP34.5 gene and an ICP47 gene of a wild II type herpes simplex virus HG52 strain are removed in the virus vector, and preferably a human granulocyte macrophage-colony stimulating factor (hGM-CSF) expression box is inserted into the position where the ICP34.5 gene is removed. The invention also provides a preparation method of the recombinant II type herpes simplex virus vector, a recombinant virus using the recombinant II type herpes simplex virus as a vector, a medicinal composition consisting of the recombinant II type herpes simplex virus vector and a pharmaceutically acceptable vector or excipient, and application of the recombinant II type herpes simplex virus vector in preparation of a gene medicament for treating tumors. As the ICP34.5 gene is removed in the recombinant II type herpes simplex virus vector provided by the invention, the oncolysis virus is safe and can selectively grow and propagate in tumor cells; the ICP47 gene is removed to promote immune response and enhance oncolysis activity; and the curative effect of the recombinant II type herpes simplex virus vector is superior to that of the conventional recombinant I type herpes simplex virus vector, and the recombinant II type herpes simplex virus vector has high safety.

Description

technical field [0001] The present invention relates to a recombinant type II herpes simplex virus vector and a preparation method thereof, a recombinant virus comprising the recombinant type II herpes simplex virus vector, a pharmaceutical composition comprising the recombinant type II herpes simplex virus vector and the recombinant type II herpes simplex virus vector And the application of recombinant virus in the preparation of gene medicine for treating tumors. Background technique [0002] Herpes simplex virus (HSV) is a double-stranded DNA virus with a length of about 154kb, which can replicate in the nucleus of infected host cells. The HSV vector has the following advantages: 1) wide range of host cells; 2) high virus titer; 3) large foreign gene capacity. A disadvantage of the HSV vector is its toxicity. [0003] For example, the ICP47 protein affects the function of the transport proteins (TAP1 and TAP2) associated with antigen processing and hinders the antigen p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/869C12N7/01A61K48/00A61K35/76A61K9/08A61P35/00A61K35/763
Inventor 刘滨磊
Owner WUHAN BINHUI BIOTECH CO LTD
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