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Method for expressing truncated non-structural protein NS4B of Tembusu virus and product and application thereof

A tambusu virus, non-structural protein technology, applied in the direction of anti-viral immunoglobulin, virus, virus peptide, etc., can solve the problem that the function and localization of non-structural protein NS4B have not been studied in detail, and achieve high sensitivity and easy Expression and ease of purification

Active Publication Date: 2018-09-04
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Tembusu virus non-structural protein NS4B plays an important role in virus gene replication and virus assembly, but so far, the function and localization of non-structural protein NS4B have not been studied in detail, and the use of tagged antibodies does not matter There are great limitations in the study of localization or its function, and the full-length NS4B protein is a transmembrane protein with multiple transmembrane regions and contains many rare codons, so we provide a truncation scheme to facilitate protein identification Expression, and the antibody prepared at the same time can recognize the full-length NS4B protein with high specificity and high sensitivity. The polyclonal antibody of NS4B plays an important role in detecting the localization of NS4B during DTMUV infection

Method used

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  • Method for expressing truncated non-structural protein NS4B of Tembusu virus and product and application thereof
  • Method for expressing truncated non-structural protein NS4B of Tembusu virus and product and application thereof
  • Method for expressing truncated non-structural protein NS4B of Tembusu virus and product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the cloning of truncated NS4B gene

[0026] 1. Proliferation culture of TMUV

[0027] The TMUV-CQW1 seed virus was inoculated into the dense monolayer duck embryo fibroblasts (DEF) that had just grown, discarded the virus solution after adsorption at 37°C for 1 hour, and then added calf serum with a volume fraction of 2% and 100IU / mL double Anti-(penicillin and streptomycin) DMEM was used to maintain the nutrient solution, and then cultured at 37°C for 24-48 hours.

[0028] 2. Extraction of total cellular RNA

[0029] 1) Select DEF (100mL cell bottle) with a cytopathic effect (CPE) of 70% after TMUV seed virus infection;

[0030] 2) Pour off the cell culture medium, add 1ml RNAiso TM Plus resuspend the cells, and react at room temperature (18-25°C) for several minutes;

[0031] 3) Add 100 μl BCP to each sample, mix manually for 30 seconds, place on ice for 10 minutes, and centrifuge at 12,000 g for 15 minutes at 4°C;

[0032] 4) Carefully pipette the ...

Embodiment 2

[0055] Example 2, pGEX-4T-NS4B 35-120aa Construction of recombinant prokaryotic expression plasmids

[0056] Double enzyme digestion pGEX-4T-1 empty plasmid, the enzyme digestion system is as follows:

[0057]

[0058] After reacting at 37°C for 15 min, linear DNA fragments were recovered with TIANGEN DNA Purification and Recovery Kit.

[0059] use II One Step Cloning Kit kit, connect the purified truncated NS4B gene fragment to the pGEX-4T-1 empty plasmid after double digestion. The reaction system is as follows:

[0060]

[0061] Note: Optimum amount of cloning vector used = [0.02 base pairs of cloning vector] ng (0.03pmol)

[0062] Optimum amount of inserts used = [0.04 base pairs of inserts] ng (0.06pmol)

[0063] After the reaction system was reacted at 37°C for 30 minutes, it was placed on ice. Take 10 μl of the ligation product and transform it into 100 μl of competent cells of the expression strain BL21(DE3), pick positive clones on the Amp plate for expan...

Embodiment 3

[0064] Example 3, Induced expression of TMUV truncated NS4B gene recombinant prokaryotic expression protein

[0065] Transform the correctly identified recombinant plasmid into BL21(DE3) host expression bacteria, pick a single clone and inoculate it into LB liquid medium containing 50 μg / mL Amp for overnight culture, and inoculate the bacterial solution shaken overnight in a new medium containing 50 μg / mL Amp the next day. In the LB medium of mLAmp, make the culture solution A600=0.05 after adding the bacterial liquid, continue to cultivate to A600≈0.6, add isopropyl-B-D-thiogalactopyranose ( IPTG) was induced to express for 10 hours, and the cells were collected by centrifugation. Add 20 mM Tris-HCL (pH=8) to resuspend the bacteria according to one-tenth of the original LB amount. Take 100 μL of the resuspension solution and add 20 μL of 6× protein loading buffer, heat and denature in a water bath at 100°C for 10 minutes, perform 12% SDS-PAGE gel electrophoresis, stain with ...

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Abstract

The invention relates to a method for expressing a truncated non-structural protein NS4B of Tembusu virus and a product and application thereof. According to the method, the non-structural protein NS4B is truncated and the truncated protein is recombined and expressed so that the truncated protein is easily expressed and the expression quantity is high. In the expression, the recombinant protein is expressed in inclusion bodies by optimizing the expression conditions, which is convenient for purification. The purified protein has immunogenicity, the multi-anti-serum with high titer can be obtained after animal immunization and the prepared antibody has high sensitivity and can be used for detection of the truncated non-structural protein NS4B of Tembusu virus and detection of localizationchange of the non-structural protein NS4B of Tembusu virus in cells in different stages and thus the method is important for the study of the Tembusu virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for expressing a non-structural protein NS4B truncated protein of Tambusu virus, and also relates to the expressed truncated protein and its application in preparing polyclonal antibodies. Background technique [0002] Tembusu virus disease (Tembusu virus disease) is an acute infectious disease caused by Tembusu virus (Tembusu virus, TMUV). Ovarian inflammation and neurological symptoms, the main clinical symptoms are loss of appetite or abolition, a sharp drop in body weight and a drop in egg production. With the continuous expansion of poultry industry in my country, Tambusu virus, as one of the main viruses threatening poultry industry in my country, has caused huge economic losses. However, there is no specific drug and treatment plan suitable for the treatment of Tembusu virus infection. [0003] Tembusu virus non-structural protein NS4B plays an important role in virus...

Claims

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Application Information

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IPC IPC(8): C07K14/18C12N15/70C07K16/10G01N33/569
CPCC07K14/005C07K16/1081C12N15/70C12N2770/24122G01N33/56983G01N2333/185
Inventor 陈舜姜博文张伟程安春汪铭书
Owner SICHUAN AGRI UNIV
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