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Recombinant 1 type herpes simplex virus and its preparation method and uses

A herpes simplex virus and virus technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as inability to detect intuitively, complex preparation methods, etc., and achieve strong targeting and good safety Effect

Inactive Publication Date: 2005-01-26
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, their preparation methods are complicated and cannot be detected intuitively.

Method used

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  • Recombinant 1 type herpes simplex virus and its preparation method and uses
  • Recombinant 1 type herpes simplex virus and its preparation method and uses
  • Recombinant 1 type herpes simplex virus and its preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction and acquisition of recombinant virus rHSV-1Δ34.5 / lacZ

[0055] The plasmid vector pSV-β-Galactosidase was purchased from Promega Company, and the transposable vector pFastBac1 was purchased from Gibco Company. The vector pSV-β-Galactosidase contains a lacZ expression cassette, and the SV40 promoter controls its expression. Plasmid vector pBluescript-M13 was purchased from Stratagene Company. Plasmid p4789 was donated by Professor Roizman B of the University of Chicago and contains the complete HSV icp34.5 gene and its flanking sequences. The Syrian hamster kidney cell line BHK21 was donated by researcher Gong Zhenkui of Hubei Academy of Medical Sciences, and the wild virus HSV-1 F strain was donated by Professor B. Roizman of the University of Chicago.

[0056] Construction of the intermediate vector IpFL: from the vector pSV-β-Galactosidase containing the lacZ expression cassette, it was completely digested with BamHI, EcoRI was incompletely di...

Embodiment 2

[0061] Example 2 Identification of recombinant virus rHSV-1Δ34.5 / lacZ

[0062] The tested viruses were the recombinant virus rHSV-1Δ34.5 / lacZ constructed in Example 1 and the wild virus HSV-1 F strain donated by Professor B. Roizman of the University of Chicago. icp34.5 gene primers were synthesized by Shanghai Sangong (SANGON) Company.

[0063] Forward primer: 5'-CTCTGCAGTCACGCCCCTTCCGCCTTCC-3'

[0064] Reverse primer: 5'-CCGGATCCGCTCCTGCCATCGTCTCTCC-3'

[0065] PCR technology was used to amplify icp34.5 gene for identification. Reaction system: 10×buffer 5μL, dNTP (2.5mM) 5μL, forward primer (10pmol / ul) 2.5μL, reverse primer (10pmol / ul) 2.5μL, HSV DNA 5μL, MgCl 2 (25mM) 5μL, ddH 2 O 25 μL, Taq DNA polymerase (5U / ul) 0.5 μL. Amplification conditions: 94°C, 90 seconds; 56°C, 90 seconds; 72°C, 120 seconds; cycle 35 times. Pre-denaturation for 5 minutes (94°C), and final extension for 10 minutes (72°C). The results proved that the recombinant virus rHSV-1Δ34.5 / lacZ delete...

Embodiment 3

[0066] Example 3 Amplification of recombinant virus rHSV-1Δ34.5 / lacZ

[0067] The test virus is the recombinant virus rHSV-1Δ34.5 / lacZ constructed in Example 1. The African green monkey kidney Vero cell line was donated by Professor Dong Changyuan from Wuhan University School of Medicine.

[0068]Use DMEM culture medium to culture Vero cells on a 35mm culture plate. After the Vero cells grow into a single layer, discard the DMEM culture medium, wash the cell surface with PBS, inoculate 0.2ml of virus, absorb at 37°C for 1-2 hours, and discard the upper layer. At the same time, set a bottle of cells without virus as a control; add 2% calf serum medium, and culture at 37°C; after 80% of the cells have lesions, freeze and thaw three times to harvest the virus; centrifuge at 8000rpm at 4°C After 30 minutes, collect the supernatant and store at 4°C for short-term storage; if long-term storage is required, add 10% glycerol and store at -80°C.

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Abstract

A recombined type 1 herpes simplex virus (Simplexvirus Herps simplexvirus 1rHSV-1Delta34.5 / lacZ), its preparation method and the application in antineoplastic are disclosed. The preparation process is: obtaining the beta-galactosidase expression box and purifying, constructing medium carrier IpFL, constructing medium carrier II pBL, constructing transfer carrier pIL and obtaining recombined virus. The apoptosis inhibiting gene icp34.5, a neurotoxic gene, is inactivateed by the reporter gene lacZ insertion. The recombined type 1 herpes simplex virus can only be replicated in the tumor cell, therefore, it is capable of killing tumor cell effectively, being safe for normal cell and tissue.

Description

Technical field: [0001] The invention belongs to the field of molecular virus genetic engineering. Specifically, the present invention relates to a recombinant type 1 herpes simplex virus, a method for preparing the recombinant type 1 herpes simplex virus, and an anti-tumor application of the recombinant type 1 herpes simplex virus. Background technique: [0002] Viruses infect sensitive host cells and replicate massively within them, eventually leading to cell lysis and death. If the virus replicates selectively in tumor cells without harming normal cells, the virus may be a highly effective tumor-killing agent. Fighting poison with fire is a classic strategy of Chinese medicine. With the development of virology, oncology, cell and molecular biology theory and technological progress, it has become possible to use genetic engineering to modify viruses to selectively kill tumor cells. [0003] There are apoptosis-inducing genes and apoptosis-inhibiting genes in many viral ...

Claims

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Application Information

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IPC IPC(8): A61K35/763A61P35/00C12N7/00C12N7/01C12N7/04C12N15/63C12N15/869
Inventor 齐义鹏董长垣肖庚富兰萍祝华斌薛峰苏越胡宝利肖巍
Owner WUHAN UNIV
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