Recombinant herpes simplex virus, preparation method and application thereof, and construction method and application of recombinant vector

A herpes simplex virus, recombinant vector technology, applied in the field of genetic engineering, can solve problems such as poor safety

Inactive Publication Date: 2019-06-11
SHENZHEN GENTARGET BIOTHERAPEUTICS CO LTD
View PDF5 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional HSV-1 has strong toxicity, not only has a killing e

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, preparation method and application thereof, and construction method and application of recombinant vector
  • Recombinant herpes simplex virus, preparation method and application thereof, and construction method and application of recombinant vector
  • Recombinant herpes simplex virus, preparation method and application thereof, and construction method and application of recombinant vector

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0063] Specifically, the preparation method of the recombinant herpes simplex virus includes the following steps S110-S150:

[0064] S110. Use the p galk vector as a template, and use the first homology arm primer pair to perform PCR amplification to obtain the ΔICP34.5-galk fragment, transfer the ΔICP34.5-galk fragment into the competent first recombinant bacteria, and perform galk After positive screening, the second recombinant bacteria were obtained. The ΔICP34.5-galk fragment contained the sequence of the upstream flank region of the ICP34.5 gene, the galk expression cassette, and the sequence of the downstream flank region of the ICP34.5 gene connected in sequence. The first recombinant bacteria contained HSV- 1. The carrier of the viral genome, the second recombinant bacteria has deleted the ICP34.5 gene, and a ΔICP34.5-galk fragment is inserted into the site of the deleted ICP34.5 gene.

[0065]The galK gene has bidirectional functions in Escherichia coli: it uses gala...

Embodiment 1

[0118] Construction of pGA1-hsPD-1 shuttle plasmid (recombinant shuttle plasmid) and detection of expression activity

[0119] (1) Synthesize hPD-1 extracellular segment sequence (hsPD-1), with a HindIII restriction site at the 5' end and an XbaI restriction site at the 3' end. The sequence of hsPD-1 is shown in SEQ ID No.1.

[0120] (2) The hsPD-1 gene was connected to the pGA1 vector with HindⅢ and XbaI restriction sites to construct the pGA1-hsPD-1 shuttle plasmid. The 5' end of the hsPD-1 gene in the pGA1-hsPD-1 shuttle plasmid is the CMV promoter, and the 3' end is BGHpA. Schematic diagram of the structure of the pGA1-hsPD-1 shuttle plasmid figure 2 shown. figure 2 Schematic diagram of the structure of the pGA1-hsPD-1 shuttle plasmid.

[0121] (3) PCR identification of pGA1-hsPD-1 shuttle plasmid

[0122] The primers in Table 1 and Phusion high-fidelity DNA polymerase (purchased from Thermo Company) were used to perform PCR reaction on the pGA1-hsPD-1 shuttle plasm...

Embodiment 2

[0134] Construction of HSV-1-ΔICP34.5-hsPD-1 recombinant plasmid

[0135] (1) Take the p galk plasmid (the schematic diagram of the structure of the p galk plasmid is shown in Figure 6 (shown) is a template, PCR amplification is performed using PrimeSTARHS DNA polymerase (purchased from TAKARA Company) and the first homology arm primer pair in Table 3 to obtain a ΔICP34.5-galk fragment. The ΔICP34.5-galk fragment contains the sequence of the flanking region upstream of the ICP34.5 gene, the galk expression cassette, and the flanking region sequence downstream of the ICP34.5 gene connected in sequence. in, Figure 6 Among them, "galk" indicates the galk expression cassette, "galkpromoter" indicates the promoter of the galk expression cassette, "kanamycin resistance" indicates the kanamycin resistance gene, "ampicillin resistance" indicates the ampicillin resistance gene, and "ampicillin promoter" indicates Promoter of the ampicillin resistance gene. The first homology arm p...

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

PropertyMeasurementUnit
Virus titeraaaaaaaaaa
Login to view more

Abstract

The invention relates to a recombinant herpes simplex virus, a preparation method and application thereof, and a construction method and application of a recombinant vector. The recombinant herpes simplex virus lacks an ICP34.5 gene and an ICP47 gene, and soluble PD-1 fragments are inserted into a site of the lacked ICP34.5 gene and a site of the lacked ICP47 gene. The recombinant herpes simplex virus is good in safety.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant herpes simplex virus, a recombinant vector, a preparation method and application thereof, a construction method of the recombinant vector and the application thereof. Background technique [0002] Programmed death receptor 1 (PD-1) is mainly expressed in activated T cells. It binds to the corresponding receptor PD-L1 or PD-L2 and transmits negative regulatory signals, leading to the shutdown of the immune response of T cells. A variety of tumor cells highly express PD-L1, and these tumor cells bind to PD-1 on the surface of T cells through the expressed PD-L1, inducing tumor-specific T cell apoptosis and immune incompetence, and eventually leading to tumor cells escaping T cells immune surveillance and killing. [0003] Oncolytic virus is a kind of tumor-killing virus with replication ability, which can reproduce in large numbers in tumor cells and kill tumor cel...

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): C12N7/01C12N15/90C12N15/85C12N15/12A61K35/763A61K48/00A61K38/17A61P35/00C12R1/93
Inventor 易吉辉孙锦霞袁文娟许春莲
Owner SHENZHEN GENTARGET BIOTHERAPEUTICS CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap