Moloney murine leukemia virus reverse transcriptase mutant as well as expression method and application thereof
A technology of murine leukemia virus and reverse transcriptase, which is applied in the field of genetic engineering and can solve problems such as the difficulty of enzyme transformation
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0019] Example 1: Construction and structural analysis of the MMLV reverse transcriptase structural model containing template-primers and dNTPs in the active state
[0020] Construction of structural models with templates / primers and dNTPs: Currently available crystal structures of MMLV include the full-length crystal structure of MMLV containing DNA far away from the catalytic site, and the N-terminal structure of MMLV reverse transcriptase without DNA in the catalytic state Crystal structure, so there are certain difficulties in the structure-based functional engineering of enzymes. There are many studies on the structure of HIV-1 reverse transcriptase, and the crystal structure of the template / primer and dNTP triple complex in the catalytic state has been obtained. The structures of MMLV reverse transcriptase and HIV-1 reverse transcriptase are highly homologous. We combined the crystal structure of HIV-1 reverse transcriptase triple complex (including enzyme, template-prim...
Embodiment 2
[0021] Example 2: Site-directed mutation of the 196-position proline (P) of MMLV reverse transcriptase is alanine (A)
[0022] Site-directed mutation of proline (P) at position 196 of MMLV reverse transcriptase (Genebank: AF033811) to alanine (A). The method of fusion PCR was used. First design the upstream and downstream primers RTUp and RTDown of the MMLV reverse transcriptase gene:
[0023] RTUp: 5'ATGCATATGACATGGCTGTCTGATTTT 3'
[0024] RTDown: 5'ATTACTCGAGTTAGAGGAGGGTAGAGGTGTCTGGAGTC 3'
[0025] And design primers 196Up and 196Down at the mutation site:
[0026] 196Up 5’CCA CAG GGT TTC AAA AAC AGT GCC ACC CTG TTT 3’
[0027] 196Down 5’AAA CAG GGT GGC ACT GTT TTT GAA ACC CTG TGG 3’
[0028] RTUp and 196Down were used as primers and the MMLV reverse transcriptase gene fragment was used as a template to amplify the N-terminal DNA fragment. Then use RTDown and 196Up as primers to amplify the C-terminal DNA fragment. The N-terminal and C-terminal DNA fragments amplified...
Embodiment 3
[0029] Embodiment 3: Expression and purification of MMLV reverse transcriptase mutant
[0030] Cloning: The MMLV mutant reverse transcriptase gene (P196A) was purified and then digested with Nde I / Xho I and cloned into the pET-28a vector, so that the His tag was added to the MMLV reverse transcriptase gene to produce pET-28a-RTP196A expression plasmid. The recombinant expression plasmid was confirmed to be correct by sequencing. Expression: The recombinant expression plasmid was transformed into E.coli BL21. Pick a single colony and culture it at 37°C until the OD600 value is 0.6, induce with 0.2mM IPTG for 3 hours, centrifuge at 4200rpm for 35 minutes, collect the bacterial liquid, and store it at -80°C. Purification: Buffer I (50mM NaH 2 PO 4 (pH 7.8), 5% glycerol, 0.3M NaCl) to suspend the bacterial liquid, add lysozyme to a final concentration of 1 mg / ml, and incubate on ice for 30 minutes to lyse. The lysed bacteria solution was centrifuged at 35,000 rpm for 40 minut...
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