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Application of id1 protein and bmp4 protein in preparation of anti-foot-and-mouth disease medicine and anti-foot-and-mouth disease medicine

A foot-and-mouth disease and protein technology, applied in the field of molecular biology, can solve the problems of short shelf life of inactivated vaccines, scattered virus, and difficulty in timely replacement of antigens of epidemic strains.

Active Publication Date: 2020-09-04
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although traditional inactivated vaccines have made significant contributions to the effective control of foot-and-mouth disease worldwide, a large number of live viruses need to be cultivated in the vaccine production process, and there is a certain risk of spreading the virus
At the same time, like other inactivated antigens, inactivated foot-and-mouth disease vaccines cannot induce broad-spectrum and long-lasting immune protection responses, and multiple immunizations are required to maintain a good immune level in the population
More importantly, inactivated vaccines have a short shelf life
In addition, some serotype viruses are difficult to pass in cells, and it is difficult to replace the circulating strain antigens in time

Method used

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  • Application of id1 protein and bmp4 protein in preparation of anti-foot-and-mouth disease medicine and anti-foot-and-mouth disease medicine
  • Application of id1 protein and bmp4 protein in preparation of anti-foot-and-mouth disease medicine and anti-foot-and-mouth disease medicine
  • Application of id1 protein and bmp4 protein in preparation of anti-foot-and-mouth disease medicine and anti-foot-and-mouth disease medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Detection of changes in the expression level of endogenous ID1 protein in cells after infection with FMDV:

[0020] 1. Taking BHK-21 cells as an example, infect the cells with 0.01MOI FMDV.

[0021] 2. Collect the cells at each time point of 5min, 10min, 20min, 30min, 1h, 2h and 6h, and extract the total cell protein.

[0022] The specific operation of extracting the total protein of monolayer adherent cells is as follows (note the operation on ice):

[0023] 1) Pour off the culture medium, turn the cell plate upside down on the absorbent paper, and let the absorbent paper absorb the culture medium.

[0024] 2) Add 1 mL of PBS to the culture dish, scrape off the cells with a cell scraper, transfer to a 1.5 mL EP tube, centrifuge at 5000 rpm at 4°C for 10 min, discard the supernatant; repeat washing once.

[0025] 3) Add 200 μL of cell lysate containing 1% protease inhibitor to the EP tube, sonicate for 5 seconds, place on ice for 20 minutes, and centrifuge ...

Embodiment 2

[0032] Example 2: Construction of stably overexpressed ID1 cell line and virus infection experiment

[0033] 1. Primer design: Download the ID1 gene sequence (NW_004801816.1 and XM_021223030) from the GenBank database, design primers for amplifying the full-length ID1 sequence, and send it to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. The primer sequence is as follows:

[0034] Forward primer SEQ ID NO: 1: cgcGGATCCatgaaggtcgccagtggtagca

[0035] Reverse primer SEQ ID NO: 2: acgcGTCGACtcagcgacacaagatgcgat

[0036] 2. Obtain the target fragment: use TRIzol to extract the RNA of BHK-21 cells, and perform reverse transcription according to the instructions of the Takara reverse transcription kit to obtain cDNA. PCR amplification was performed using the obtained cDNA as a template, and the amplified product was subjected to agarose gel electrophoresis, recovered and purified by cutting the gel, digested with BamHI and SalI, and ligated into the pCMV-Flag vector.

[0...

Embodiment 3

[0061] Example 3: BMP4 inhibits FMDV replication by inducing high expression of ID1

[0062] 1. 60mm dish middle shop 2.6*10 6 BHK-21 cells were treated with BMP4 at a final concentration of 10 ng / mL when the confluence reached 90%, and the cells were collected 2 hours and 12 hours after treatment to extract cell proteins. The protein extraction method was the same as above, and the expression of ID1 was detected by Western blot.

[0063] The result is as image 3 As shown in the middle panel a, BMP4 can effectively up-regulate the expression of ID1. Ctrl is the untreated BHK-21 cell sample, and the rest are the cell samples collected 2h and 12h after BMP4 treatment.

[0064] 2. 60mm dish middle shop 2.6*10 6 BHK-21 cells were pretreated with BMP4 at a final concentration of 10 ng / mL when the confluence reached 90%, infected with 0.01 MOI of FMDV 2 hours later, and collected cell samples 8 hours later to extract RNA. The method of extracting total cellular RNA was the sam...

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Abstract

The invention relates to the field of molecular biology, and discloses an anti-foot-and-mouth disease drug and an application of ID1 protein and BMP4 (bone morphogenetic protein 4) protein in preparation of the same. An embodiment of the invention initially discover the relation of ID1 protein and the replication of the foot-and-mouth disease, which includes that the cure of the foot-and-mouth disease is realized through upregulating the expression of Id1. According to the function of the ID1 protein, the ID1 protein can be utilized in the preparation of the anti foot-and-mouth disease drug, and an important theoretical basis is provided in the development of new anti foot-and-mouth disease drug.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of ID1 protein and BMP4 protein in the preparation of anti-foot-and-mouth disease medicine and the anti-foot-and-mouth disease medicine. Background technique [0002] Foot and mouth disease (FMD) is an acute, febrile, highly contagious disease caused by foot and mouth disease virus (FMDV). The World Organization for Animal Health (OIE) lists foot and mouth disease as a must-report Number one animal disease. The prevention and control of the disease in my country and other countries mainly adopts the preventive measures of vaccine immunization. Although traditional inactivated vaccines have made great contributions to the effective control of foot-and-mouth disease worldwide, a large number of live viruses need to be cultivated in the vaccine production process, and there is a certain risk of spreading the virus. At the same time, like other inactivated antigens,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K38/18A61P31/14
CPCA61K38/1709A61K38/1875A61P31/14A61K2300/00
Inventor 孙跃峰张永光任亭亭祁林林陈豪泰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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