Enhanced subsample gene capable of improving expression of foreign protein and application thereof
An enhancer-like, exogenous protein technology, applied in the direction of using vectors to introduce foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of undiscovered enhancer-like sequences, so as to increase the possibility and increase The effect of practicality
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0034] Example 1: Construction of pET21a-CAT-L11 enhancer-like sequence detection vector
[0035] The tumor tissue of the cervical cancer patient in the First People's Hospital of Yunnan Province was taken, and the genome of the sample was extracted according to the DNA extraction method of animal tissue genome in the "Guidelines for Molecular Biology Experiments" (Science Press). After identification, the sample was HPV58 type Infection, HPV58 type L1 was amplified by PCR using primers L11P1: 5'-GGA ATT CCA TAT GTC TGT TTG GAG ACC TTC-3'; L11P2: 5'-CCG CTC AGA CAG TGA GTC TCC ATA AGG TTC-3' The truncated sequence L11 of the gene was constructed into a pMD18T-L11 recombinant plasmid (the pMD18T vector was purchased from TAKARA Company, CK5601C), and the plasmid was transformed into Escherichia coli DH5α (DH5α was purchased from TAKARA Company, D9057), and the recombinant plasmid was cultured with shaking at 37°C Bacteria pMD18T-L11 / DH5α, after 16 hours, use the plasmid mini-...
Embodiment 2
[0038] Example 2: Detection of Recombinant Bacteria Chloramphenicol Resistance Threshold and L11-CAT Fusion Protein Expression Level
[0039] Take 10ng of the enhancer-like sequence detection plasmid pET21a-CAT-L11 and 100 μL E.coli BL21 (DE3) (purchased from Novagen, 69450-3) and mix competently, bathe in ice water for 30 minutes, and heat shock in a warm water bath at 42°C for 2 minutes. Then add 1ml of LB culture solution, shake and culture at 37°C for 45 minutes, discard most of the supernatant after centrifugation and resuspend the bacteria, and spread on Amp + -In LB solid petri dish, cultivate at 37°C for 12-16h to obtain the recombinant screening strain pET21a-CAT-L11 / BL21 (DE3); pick the single colony and inoculate it into Amp + -In LB liquid medium, shake culture overnight at 37°C, then inoculate Amp at a ratio of 1% + -LB liquid medium, cultured with shaking at 37°C until OD 600 About 0.5, 10 for the bacterial solution 5 times and 10 6 After doubling dilution,...
Embodiment 3
[0041] Example 3: Genome extraction, digestion and library construction
[0042] After shaking and mixing, 150μL was added to 15ml beef extract peptone liquid medium, and shake cultured at 37°C. After 16 hours, divide the culture solution into 10ml centrifuge tubes, centrifuge at 12000g×2min, discard the supernatant, and resuspend the bacteria with 2ml TE buffer, add 180μL of 10% SDS and 12μL of 20mg / ml proteinase K, and mix well at 37 Incubate at ℃ for 1 h, then add 600 μL of 5M NaCl and mix well, add 400 μL of CTAB / NaCl solution, incubate at 65 °C for 10 min; add an equal volume of chloroform, mix well, centrifuge at 12000 g×5 min, take the supernatant, add etc. Mix the volume of phenol / chloroform mixture evenly, centrifuge at 12000g×5min, take the supernatant and distribute it into 1.5ml centrifuge tubes, 700μL / tube, add an equal volume of isopropanol, invert and mix well, store at -40℃ Let stand for 30min, centrifuge at 12000g×10min, discard the supernatant, and then ri...
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