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

A kind of porcine ifitm3 gene that inhibits the proliferation of foot-and-mouth disease virus and its construction method and application

A foot-and-mouth disease virus and gene technology, which is applied in the field of construction of pig IFITM3 gene, can solve the problems of difficulty in epidemic prevention work, increase the complexity of epidemic situation and control difficulty, etc.

Inactive Publication Date: 2011-12-21
HUAZHONG AGRI UNIV
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is no obvious cross-protection between the various subtypes of foot-and-mouth disease virus, the complexity of the epidemic and the difficulty of control have increased, making the epidemic prevention work very difficult

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
  • A kind of porcine ifitm3 gene that inhibits the proliferation of foot-and-mouth disease virus and its construction method and application
  • A kind of porcine ifitm3 gene that inhibits the proliferation of foot-and-mouth disease virus and its construction method and application
  • A kind of porcine ifitm3 gene that inhibits the proliferation of foot-and-mouth disease virus and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: a kind of preparation method of the porcine IFITM3 gene that suppresses foot-and-mouth disease virus propagation, its step is:

[0067] 1. Cloning of porcine IFITM3 gene (sIFITM3) and construction of eukaryotic vector pCA-sIFITM3

[0068] Referring to GenBank human and mouse IFITM gene sequences, compared with pig EST sequences, selected the sequence with the highest homology as the target sequence, designed a pair of primers, extracted pig lymph node tissue RNA, and performed RT-PCR to amplify the sIFITM3 gene , the primer sequences are as follows:

[0069] sIFITM 3F: 5'-tttgaattccaccatgaactgcgct-3'

[0070] sIFITM 3R: 5'-tttctcgagtcagatcatcgga-3'

[0071] A two-step method was used for amplification. Firstly, cDNA was obtained by reverse transcription, and then used as a template for PCR amplification. The reaction system was: template plasmid 0.25 μL, 10×LAbuffer 5 μL, pTRIM25F and pTRIM25R primers 1.0 μL each (final 10 pmol / L), LA enzyme 0.25 μL, dN...

Embodiment 2

[0086] Example 2: Application of a porcine IFITM3 gene, cell line BHK21-sIFITM3eGFP, lentivirus Lentivirus-sIFITM3 and eukaryotic plasmid pCA-sIFITM3 that inhibit the proliferation of foot-and-mouth disease virus in the preparation of drugs for treating or preventing foot-and-mouth disease virus. (Are these five kinds of application processes the same? If they are the same, they can be described in the same embodiment, and if they are different, describe the applications separately?

[0087] 1, a kind of cell line BHK21-sIFITM3eGFP of the porcine IFITM3 gene that suppresses foot-and-mouth disease virus propagation is in the application in the preparation treatment or prevention foot-and-mouth disease virus medicine, and its steps are:

[0088] ① Plaque test: In order to initially observe the effect of sIFITM3 protein on the proliferation of FMDV, the same number of BHK21-sIFITM3eGFP and BHK-21 cells were inoculated into a 6-well plate, and the plaque test was performed after th...

Embodiment 3

[0097] Embodiment 3: the application of a kind of sIFITM3 gene that suppresses foot-and-mouth disease virus proliferation in transgenic animals such as transgenic pigs, its application process is:

[0098] In order to evaluate the potential breeding value of sIFITM3 gene, the antiviral activity of sIFITM3 gene was studied at the body level. The eukaryotic plasmid pCA-sIFITM3 was digested with AccI and HindIII, and the 2606bp fragment containing the promoter, sIFITM3 and polyA was recovered in TE for transgene, with a final concentration of 2 μg / ml. Then microinjection was carried out by conventional methods (Yu Zheng. Research on the expression of recombinant lysozyme with antibacterial activity in the mammary gland of transgenic mice, China Agricultural University, doctoral dissertation) for the research of transgenic mice / pigs. The tail DNA of the obtained mice was extracted, and PCR amplification was performed with sIFITM 3F (5'-tttgaattccaccatgaactgcgct-3') and sIFITM 3R (...

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 pig IFITM3 gene for inhibiting mouth disease virus multiplication, and a construction method and an application. The method comprises the following steps of: A, synthesizing pig IFITM3 genes: designing primers, extracting pig lymphoid tissue RNA, carrying out RT-PCR multiplication on sIFITM3 genes, carrying out multiplication, using pCA-sIFITM3 for realizing transfectionon BHK-21 cells and observing the protein to be expressed on the cell membrane through a confocal microscopy; B, constructing and purifying the slow virus for expressing the sIFITM3 genes; and C, constructing the BHK21-sIFITM3eGFP cell systems for stably expressing the sIFITM3 genes: constructing the BHK-21 cell systems for stabilizing the sIFITM3 genes, using Lentivirus-sIFITM3eGFP and LentiviruseGFP for respectively realizing the transfection on the BHK-21 cells, carrying out cloning screening by green fluorescence and identifying the expression of the sIFITM3 being BHK21-sIFITM3eGFP through RT-PCR and Westernblotting. The invention also discloses the application of the gene, the cell system BHK21-sIFITM3eGFP, the slow virus Lentivirus-sIFITM3 and the eukaryotic plasmid pCA-sIFITM3 to the preparation of medicine for treating or preventing the mouth disease virus. The constructed BHK21-sIFITM3eGFP cell systems can stably express sIFITM3 protein, BHK21-sIFITM3eGFP can inhibit the copying of the mouth disease virus, and the slow virus lentivirus-sIFITM3 carrying sIFITM3 and the eukaryotic plasmid pCA-sIFITM3 can inhibit the copying of the mouth disease virus at the organism level.

Description

technical field [0001] The invention relates to the fields of virology and disease resistance breeding. More specifically, it relates to a pig IFITM3 gene (interferon induced transmembrane 3, IFITM3) that inhibits the proliferation of foot-and-mouth disease virus, and also relates to a method for constructing a pig IFITM3 gene that inhibits the proliferation of foot-and-mouth disease virus, and also relates to a pig IFITM3 gene that inhibits the proliferation of foot-and-mouth disease virus. Gene application in foot-and-mouth disease resistance breeding. Background technique [0002] my country is the largest pig raising country in the world. Pig farming is the leading industry of my country's animal husbandry, pork accounts for 67.7% of meat, and is the most important source of animal protein for our people. Infectious diseases seriously endanger human and animal health. Every year, 15 million people die from infectious diseases in the world. In my country, 11.6 million ...

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): C12N15/10C12N7/01C12N5/10A61K35/23A61K48/00A61K38/17A61P31/14A01K67/027A61K35/22
Inventor 钱平李祥敏许晋芳刘莎莎周锐金梅林陈焕春
Owner HUAZHONG AGRI UNIV
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