Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP and corresponding diagnostic kit thereof

A herpes simplex virus, virus genome technology, applied in the direction of virus, virus/phage, double-stranded DNA virus, etc., can solve the problem of low virus reproduction titer

Active Publication Date: 2017-02-22
重庆宇珩生物科技有限公司
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims to solve the defect of low reproductive titer of existing viruses, and provides a recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP, which has selective high-titer fecundity, and the virus has a stable reproductive titer in liver cancer cells. 10 6 above

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP and corresponding diagnostic kit thereof
  • Recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP and corresponding diagnostic kit thereof
  • Recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP and corresponding diagnostic kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] A preparation method for recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP, comprising the following steps:

[0085] 1) Replace the ICP4 gene promoter in the herpes simplex virus containing the ICP4 gene with the human telomerase reverse transcriptase promoter hTERTp to construct the recombinant herpes simplex virus HSV-hTERTp_ICP4:

[0086](1) Construction of shuttle plasmids pICP4del-hTERTp_ICP4 and pICP4del-eGFP

[0087] a. Cultivate the herpes simplex virus containing ICP4 gene with BHK cells, and purify its genomic DNA;

[0088] b. Amplify the flanking sequence upstream of the ICP4 gene: with the viral genome DNA obtained in step a) as a template, use the following ICP4USf forward primer and ICP4USr reverse primer:

[0089] ICP4USf forward primer: ccctccagacgcaccggagtcggggg,

[0090] ICP4USr reverse primer: aagtcgactctagaggatcgatctctgacctgagattggcggcactgaggta

[0091] Amplify the upstream flanking sequence of the ICP4 gene;

[0092] c. Amplify the downst...

Embodiment 2

[0143] Embodiment 2HSV_Hep-GFP selectively propagates and produces high-titer virus in liver cancer cells:

[0144] HSV_HEP-GFP was used to infect liver cancer cells (HuH7, HepG-2, 7721), lung cancer cells (A549, PG), gastric cancer cells BGC823, colon cancer cells HT-29, human erythroleukemia cells TF-1, lymph Tumor cell U937, breast cancer cell MD-MB-231, pharyngeal squamous cell carcinoma cell Fadu, and melanoma cell A375 were tested for virus titer at 6, 12, 24, 48, and 72 hours. The results are shown in Table 1. Experimental data show that HSV_HEP-GFP has the best effect on infecting liver cancer cells, and the virus reproduction titer after 24 hours or later is significantly higher (more than 2 logarithms) than that of other cancer cells that can reproduce the virus.

[0145]

Embodiment 3

[0146] Embodiment 3HSV_Hep-GFP produces the virus of higher titer than HSV_ICP4 in liver cancer cell

[0147] HSV_HEP and HSV_ICP4 were respectively infected with liver cancer cells HuH7, HepG-2, and 7721 at MOI=0.01, and the virus titers were detected at 6, 12, 24, 48, and 72 hours. see results figure 1 -3. Experimental data showed that HSV_HEP infected three kinds of liver cancer cells, and the viral reproduction titers reached 10 after 24 hours. 6 Above, although HSV_ICP4 can reproduce in three kinds of liver cancer cells, but the virus titer is low, the peak value is only 10 4 . The two viruses infected liver cancer cells, and their reproductive titers differed by 2 logarithms.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP and a corresponding diagnostic kit thereof. A 25bp DNA segment is inserted between a telomerase reverse transcriptase promoter and an ICP4 gene on the virus genome, and a GFP (green fluorescent protein) expression cassette is inserted in the ICP34.5 site, wherein the sequence of the DNA segment is TTGCCCCAAGCGGCATTTGGGTTCA. The virus has optionally high titer fertility, and the liver cancer cell reproduction titer of the virus is stably higher than 10<6>. The synthetic random nucleotide short sequence and HSV1-hTERTp_ICP4 virus genome are subjected to site-specific recombination (between hTERTp and ICP4). The change of the short sequence can increase the virus reproduction titer and enable different viruses to generate optionally high titer reproduction according to different types of tumor cells, i.e., some viruses can reproduce better in liver cancer cells, but some viruses can generate higher titer in liver cancer cells.

Description

technical field [0001] The invention relates to a recombinant herpes simplex virus, in particular to a recombinant herpes simplex virus HSV-hTERTp_ICP4_Hep-GFP and a corresponding diagnostic kit. Background technique [0002] Telomere is a special structure at the end of eukaryotic chromosomes. Its function is to maintain the stability of chromosome structure, including preventing fusion of chromosome ends, protecting chromosome structure genes and avoiding loss of genetic information during replication. Telomerase is a reverse transcriptase composed of small molecule RNA and protein, which can use its own RNA as a template to synthesize telomere DNA and make up for the telomere that gradually shortens with cell mitosis. It has three main components: human telomerase RNA (human telomerase RNA, hTR), telomerase-associated protein (telomerase-associated protein, TP1 / TLP1), human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT ). Telomerase RNA...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N7/01C12N15/65C12Q1/70C12Q1/06G01N15/14C12R1/93
CPCG01N15/14G01N33/5005C12N7/00C12N15/65C12N2710/16621Y02A50/30
Inventor 谷明张婧
Owner 重庆宇珩生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com