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
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[0025] Example 1. Cloning of truncated NS4B gene
[0026] 1. Proliferation and culture of TMUV
[0027] The TMUV-CQW1 virus was inoculated into the newly grown dense monolayer duck embryo fibroblasts (DEF), adsorbed at 37°C for 1 hour, and then discarded the virus solution, and then added 2% calf serum and 100IU / mL double Anti-(penicillin and streptomycin) DMEM is maintained as a nutrient solution, and then incubated at 37°C for 24 to 48 hours.
[0028] 2. Extraction of total cell RNA
[0029] 1) Select DEF (100mL cell bottle) with 70% cytopathic changes (CPE) after TMUV seed infection;
[0030] 2) Pour out the cell culture medium and add 1ml RNAiso TM Plus resuspend the cells and react at room temperature (18~25℃) for several minutes;
[0031] 3) Add 100μl BCP to each sample, mix manually for 30s, place on ice for 10min, centrifuge at 12000g for 15min at 4℃;
[0032] 4) Carefully pipet the transparent supernatant into a new RNase-free EP tube, add 500μl of isopropanol to each tube, gen...
Example Embodiment
[0055] Example 2, pGEX-4T-NS4B 35-120aa Construction of recombinant prokaryotic expression plasmid
[0056] Double digestion pGEX-4T-1 empty plasmid, digestion system is as follows:
[0057]
[0058] After reacting for 15 minutes at 37°C, linear DNA fragments were recovered with TIANGEN DNA Purification and Recovery Kit.
[0059] use II One Step Cloning 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: Optimal cloning vector usage = [0.02 base pairs of cloning vector]ng (0.03pmol)
[0062] Optimal insert usage amount = [0.04 insert base pairs]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 to transform into 100 μl of competent cells of the expression strain BL21(DE3), pick the positive clones on the Amp plate for expansion culture, and extract the plasmid for sequencing.
Example Embodiment
[0064] Example 3. Induced expression of TMUV truncated NS4B gene recombinant prokaryotic expression protein
[0065] The identified correct recombinant plasmid was transformed into BL21(DE3) host expression bacteria, and a single clone was picked and inoculated into LB liquid medium containing 50μg / mL Amp and cultured overnight. The next day, the overnight bacterial solution was inoculated into a new strain containing 50μg / mL Amp. In mLAmp's LB medium, make the culture solution A600 = 0.05 after adding the bacterial solution. When the culture is continued to A600≈0.6, add isopropyl-BD-thiogalactopyranoglycan at the final concentration of 0.4Mm and 0.5Mm. IPTG) was induced to express for 10 hours, and the cells were collected by centrifugation. 20mM Tris-HCL (pH=8) was added to resuspend the bacteria at 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...
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