Non-structural protein gene 3ABC of foot-and-mouth disease virus and its preparation and use
A non-structural protein and foot-and-mouth disease virus technology, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, sugar derivatives, etc., can solve the problems of diffusion, live virus escape, incomplete virus inactivation, etc., and achieve biological safety High, low production cost effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0057] Cloning of cNDA Sequence of Nonstructural Protein Gene 3ABC of Foot-and-Mouth Disease Virus
[0058] The foot-and-mouth disease virus was inoculated on the passaged hamster kidney cells (BHK) covered with a single layer, and the virus was collected when 80% of the cells showed cytopathic changes. Using the extracted FMDV RNA as a template, 3ABC cDNA of FMDV nonstructural protein gene was amplified by RT-PCR. The primers used for RT-PCR are designed according to the genome sequence of FMDV reported by D.K.J Mackay et al. (Vaccine.1998,16(5):446-459), and its sequence is as follows:
[0059] P1: 5'-TGGATCCATGATTTCAATCCCTTCC-3' (upstream primer)
[0060] P2: 5'-GCGAATTCATCACTCGTGGTGTGG 3' (downstream primer)
[0061] The RT-PCR reaction system was: template RNA 8 μL, 2×Reaction buffer 25 μL, P1 and P2 primers 1.0 each (final concentration 10 pmol / L), Taq / mix 1.0 μL, add DEPC-treated water to 50 μL. The RT-PCR reaction conditions were 50□ for 30 minutes, 94□ for 1 minute; ...
Embodiment 2
[0063] Construction of Prokaryotic Expression Vector of Foot-and-Mouth Disease Virus Nonstructural Protein 3ABC Gene
[0064] Digest pT-3ABC and vector pGEX-KG with BamHI and Xba I respectively, recover 3ABC gene and vector pGEX-KG; then connect with T4 DNA ligase, 16□ water bath overnight, transform DH5α competent bacteria, culture at 37□, randomly Multiple single colonies were selected, placed in LB liquid medium and cultured at 37°C for 12 hours, and then the plasmid was extracted from it. After enzyme digestion and identification, a positive recombinant plasmid was screened and named pKG-3ABC.
Embodiment 3
[0066] Construction of recombinant Escherichia coli BL21 / pKG3ABC and Escherichia coli BL21 / pKG
[0067] Transform the recombinant expression vector pKG-3ABC into Escherichia coli BL21 competent cells, spread LB / ampicillin (Amp) plates, pick multiple single colonies and put them in LB liquid medium for 12 hours at 37°C, and then use isopropyl Thio-β-D-galactoside (IPTG) induced expression, followed by SDS-PAGE and western-blot detection, from which the recombinant Escherichiacoli BL21 capable of inducing the expression of foot-and-mouth disease virus nonstructural protein 3ABC in Escherichia coli BL21 was screened / pKG3ABC.
[0068] Simultaneously, the vector pGEX-KG was transformed into Escherichia coli BL21 cells synchronously, induced and expressed in Escherichia coli BL21 according to the above method, and a blank control recombinant Escherichia coil BL21 / pKG without expression of foot-and-mouth disease virus nonstructural protein 3ABC was screened out.
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
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