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

Kit for amplifying herpes simplex virus (HSV)-1 alkali nuclease gene and method for expressing HSV-1 alkali nuclease

A nuclease and kit technology, applied in the field of HSV-1 alkaline nuclease, can solve the problems of no alkaline nuclease, high cost, and difficulty in industrial application

Inactive Publication Date: 2011-02-23
CHENGDU MEDICAL COLLEGE
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The technical scheme of this method is complicated, and it is difficult to obtain a large amount of alkaline nuclease, the cost is high, and industrial application is difficult
There is no commercially available alkaline nuclease

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
  • Kit for amplifying herpes simplex virus (HSV)-1 alkali nuclease gene and method for expressing HSV-1 alkali nuclease
  • Kit for amplifying herpes simplex virus (HSV)-1 alkali nuclease gene and method for expressing HSV-1 alkali nuclease
  • Kit for amplifying herpes simplex virus (HSV)-1 alkali nuclease gene and method for expressing HSV-1 alkali nuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 amplifies UL12 gene fragment

[0057] (1) Virus genome extraction: culture vero cells with RPMI 1640 medium containing 10% fetal bovine serum, inoculate HSV-1 until the cells are damaged, that is, the CPE reaches +++~++++, harvest the cells, and extract them with viral DNA Extraction kit to extract the whole genome of HSV-1;

[0058] (2) According to the nucleotide sequence of the UL12 gene fragment published by GenBank, primers were designed with PRIMER5.0 software, and the primer sequences are shown in SEQ ID NO: 1-2;

[0059] (3) Amplify the target gene: take the extracted HSV-1 whole genome as a template, and use the nucleotide sequence as primer pair 1 shown in SEQ ID NO: 1~2 to amplify the target gene UL12 gene fragment:

[0060] reaction system:

[0061] Template 1μl

[0062] Nucleotide sequence as shown in SEQ ID NO: 1 Primer 1μl

[0063] Nucleotide sequence as shown in SEQ ID NO: 2 Primer 1μl

[0064] GC buffer 12.5μl

[0065] dNTP 2μl

[0...

Embodiment 2

[0071] Embodiment 2 further amplifies the UL12 gene fragment

[0072] (1) Template preparation: The UL12 gene fragment obtained in Example 1 was ligated with the pMD-19T vector under the action of T4 DNA ligase to construct a recombinant pMD19T vector.

[0073] (2) Amplify the target gene: use the recombinant pMD19T vector connected with the UL12 gene fragment obtained in step (1) as a template, and use the nucleotide sequence such as the primer pair shown in SEQ ID NO: 1-2 as primers to amplify the target gene UL12 gene fragment:

[0074] reaction system:

[0075] Template 1μl

[0076] Nucleotide sequence as shown in SEQ ID NO: 1 Primer 1μl

[0077] Nucleotide sequence as shown in SEQ ID NO: 2 Primer 1μl

[0078] GC buffer 12.5μl

[0079]2 μl of dNTPs

[0080] 0.25 μl of LAtaq

[0081] wxya 2 7.25 μl of O

[0082] Reaction conditions: pre-denaturation at 96°C for 3min, denaturation at 95°C for 40s, annealing at 68°C for 2min and 30s, 31 cycles, extension at 72°C for...

Embodiment 3

[0086] Expression of embodiment 3 alkaline nuclease and the identification of existing form

[0087] (1) Construction of recombinant expression plasmid

[0088] The UL12 gene fragment obtained in Example 1 or Example 2 is connected with pET28a (+) plasmid or pET32a (+) under the action of EcoRI and HindIII endonuclease to form pET28a-UL12 recombinant expression plasmid or pET32a-UL12 recombinant expression plasmid, The constructed recombinant plasmid contains T7 RNA polymerase promoter, controls the expression of alkaline nuclease, and contains 2 His tags, which can be used for protein purification.

[0089] (2) Recombinant expression plasmid induced expression in Escherichia coli BL21

[0090] Pass the pET28a-UL 12 recombinant expression plasmid that step (1) obtains through CaCl 2 Mediate the transformation into E.coli BL21 competent cells, culture at 37°C on LB culture plates containing 50 μg / ml karamycin, shake at 220r / min overnight;

[0091] Pick the Escherichia coli c...

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 provides a kit for amplifying a gene segment UL12 of herpes simplex virus (HSV)-1 alkali nuclease. The kit comprises a primer pair of which the nucleotide sequence is shown as SEQ ID NO:1-2 and other relevant reagents. The invention also provides a method for obtaining HSV-1 alkali nuclease with bioactivity by using the kit. A large amount of HSV-1 alkali nuclease with high bioactivity can be obtained by a genetic engineering method, the alkali nuclease play an important role in further finding an anti-virus medicament and clarifying a medicament action mechanism and an experimental basis is laid for in-vitro construction of a medicament screening molecular model of the HSV-1 alkali nuclease.

Description

technical field [0001] The invention relates to a method for obtaining viral protein, in particular to a method for amplifying HSV-1 alkaline nuclease gene by PCR technology and obtaining HSV-1 alkaline nuclease through gene expression. Background technique [0002] Herpes simplex virus type 1 (HSV-1) is an enveloped DNA virus, which is a common pathogen that endangers human health and easily causes recurrent infection and latent infection. HSV-1 has a wide range of infection sites, can cause cold sores, genital herpes, encephalitis, keratitis, etc., and is related to cervical cancer and lip cancer. HSV-1 is neurotoxic and can invade the central nervous system and destroy nerve cells. [0003] Alkaline nuclease is encoded by the UL12 gene in the HSV-1 genome. It has 5'-3' exonuclease and endonuclease activities. It is considered to be an alkali because of its high optimum pH value. sexual nuclease. It plays an important role in the replication of HSV-1. Studies have confi...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/55C12N9/22C12N15/70
Inventor 陈恬张磊曹康郭洪秀代富英
Owner CHENGDU MEDICAL COLLEGE
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