Recombined II-type herpes simplex virus, preparation method and application and tumour diagnostic reagent kit

A herpes simplex virus and virus technology, applied in the field of recombinant herpes simplex virus type Ⅱ, its preparation and application, and tumor diagnostic kits, can solve the problems of limited tumor types, low detection rate of micrometastases, and poor detection sensitivity

Active Publication Date: 2011-10-19
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI +1
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Problems solved by technology

[0005] The purpose of the present invention is to overcome the shortcomings of low detection rate of micro-metastases, poor detection sensitivity and limited types of tumors in the existing methods for early diagnosis of tumors and tumor metastasis, and to provide a recombinant type II herpes simplex virus, Early diagnosis of tumors and diagnosis of tumor metastasis can be realized quickly, accurately, sensitively and broad-spectrum by using the virus

Method used

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  • Recombined II-type herpes simplex virus, preparation method and application and tumour diagnostic reagent kit
  • Recombined II-type herpes simplex virus, preparation method and application and tumour diagnostic reagent kit
  • Recombined II-type herpes simplex virus, preparation method and application and tumour diagnostic reagent kit

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preparation example Construction

[0035] The present invention also provides a method for preparing recombinant herpes simplex virus type II, wherein the method comprises deleting the ICP34.5 gene from the wild-type herpes simplex virus type II strain HG52 to construct recombinant herpes simplex virus type II HG52d34.5- Fluorescent protein steps:

[0036] A. Extract the full-length viral DNA of the wild type II herpes simplex virus HG52 strain;

[0037] B. Construction of the plasmid pH2dI34.5 containing the upstream flanking region sequence and the downstream flanking region sequence of the ICP34.5 gene:

[0038] B1. Use the primers shown in Table 1, and use the full-length viral DNA obtained in step A as a template to amplify the upstream flanking region sequence and the downstream flanking region sequence of the ICP34.5 gene by PCR;

[0039] Table 1

[0040]

[0041] B2. Insert the upstream flanking region sequence of the PCR product amplified in step B1 into the PvuII / XbaI site of the pSP72 plasmid to...

Embodiment 1

[0102] This example is used to illustrate the recombinant type II herpes simplex virus of the present invention.

[0103] Delete the ICP47 gene from the wild-type HSV-2 (HG52) genome to construct HG52dICP47 .

[0104] (1) DNA of purified HG52 wild-type virus

[0105] BHK cells were used to grow wild-type HG52 virus, 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 ...

Embodiment 2-4

[0167] This example is used to illustrate the recombinant type II herpes simplex virus of the present invention.

[0168] According to the same principle as the steps of Example 1, the recombinant virus HG52d34.5GFP of Example 2 (the ICP34.5 gene was deleted and the GFP expression cassette was inserted at the position of the ICP34.5 gene), the recombinant virus HG52dICP47GFPd34 of Example 3 were prepared. .5 (deleting ICP34.5 gene and ICP47 gene, and inserting GFP expression cassette at the position of ICP47 gene) and embodiment 4HG52dICP47GFPd34.5GFP (deleting ICP34.5 gene and ICP47 gene, and inserting GFP expression box at the position of ICP34.5 gene and ICP47 gene Insert the GFP expression cassette at the position).

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Abstract

The invention provides recombined II-type herpes simplex virus. In the virus, ICP34.5 gene of a wild II-type herpes simplex virus HG52 strain is excluded, and a fluorescent protein expression box is inserted in the gene group. The invention also provides a preparation method of the recombined II-type herpes simplex virus, the application of the recombined II-type herpes simplex virus in preparation of the drug for diagnosing tumours, and a tumour diagnostic reagent kit with the recombined II-type herpes simplex virus. In the recombined II-type herpes simplex virus disclosed by the invention, the ICP34.5 gene is excluded, so that the recombined II-type herpes simplex virus can selectively grow and breed in tumour cells, the recombined II-type herpes simplex virus is inserted in the fluorescent protein expression box and can give out fluorescence in tumour cells, so that the tumour diagnostic reagent kit with the recombined II-type herpes simplex virus can quickly, accurately, sensitively and broadly detect whether a sample to be detected contains tumour cells or not.

Description

technical field [0001] The present invention relates to recombinant type II herpes simplex virus and its preparation method, the preparation method of said recombinant type II herpes simplex virus, the application of said recombinant type II herpes simplex virus in the preparation of drugs for diagnosing tumors and the recombinant Tumor diagnostic kit for herpes simplex virus type II. Background technique [0002] There are many deficiencies in the existing early diagnosis of tumor and tumor metastasis diagnosis technology. [0003] For example, imaging diagnosis (such as X-ray diagnostic technology, ultrasonic diagnostic technology, X-ray computed tomography (CT), endoscopic technology, magnetic resonance imaging (MRI), multi-slice spiral CT, positron emission tomography ( PET) and other technologies) have a low detection rate for micrometastases; histopathological diagnosis (such as sentinel lymph node biopsy, bone marrow biopsy, etc.) will bring great pain to the subject...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12Q1/70C12Q1/02G01N21/64C12R1/93
Inventor 刘滨磊施桂兰庄秀芬李洁张郁张叔人刘宝印张友会
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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