Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof

A technology of genetically engineered bacteria and nucleotide sequences, applied in the field of repetitive palindromic sequences, can solve the problem of limited production of cyclodextrin glucosyltransferase and achieve the effect of increasing expression

Active Publication Date: 2018-09-14
JIANGNAN UNIV
View PDF11 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is very limited to increase the yield of cyclodextrin glucosyltransferase through simple heterologous expression, and it is often necessary to further enhance its heterologous expression through molecular modification

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof
  • Repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof
  • Repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Preparation of expression vectors containing different repetitive palindromic sequences

[0026] Using the recombinant plasmid CGT-P1-1-DB3 constructed in our laboratory containing the gene sequence of cyclodextrin glucosyltransferase shown in SEQ ID NO.4 as a template, using phosphorylated PDRep1F / PDRep1R as primers, using PCR amplification of a series of recombinant plasmids containing repetitive palindromic sequences. The obtained series of recombinant plasmids were transformed into Escherichia coli host bacteria, and a series of recombinant engineered bacteria CGTaseBL21(DE3) secreting and expressing cyclodextrin glucosyltransferase were obtained. The recombinant engineering bacteria were inoculated into 96 deep-well plates, and the CGT enzyme activity was measured after 90 hours of fermentation, and 4 groups with the highest enzyme activity were screened out, and the recombinant plasmids were named CGT-REP1 / CGT-REP2 / CGT-REP3 / CGT-REP4, the repetitive pal...

Embodiment 2

[0030] Example 2 The preparation of the expression vector containing the spacer between the repetitive palindromic sequence of different lengths and the stop codon

[0031]Using the plasmid CGT-REP1 template, using spacer4F to spacer20R as primers, use PCR to amplify recombinant plasmids containing different lengths of the spacer between the repetitive palindromic sequence and the stop codon, which are named CGT-REP1-S4 / CGT -REP1-S8 / CGT-REP1-S12 / CGT-REP1-S16 / CGT-REP1-S20, the spacer sequences contained therein are respectively as SEQ ID NO.7 / SEQ ID NO.8 / SEQ ID NO.9 / SEQ Shown in ID NO.10 / SEQ ID NO.11. The above-mentioned recombinant plasmid was transformed into E. coli host bacteria, and the CGT enzyme activity was measured after 90 hours of fermentation, and the strain with the highest enzyme activity was screened out, so as to obtain the optimal spacer length between the repetitive palindromic sequence and the stop codon.

[0032] The primer sequences used in this embodiment...

Embodiment 3

[0044] Induced expression of embodiment 3 recombinant escherichia coli

[0045] Seed cultivation: transfer the preserved strains into a 250mL Erlenmeyer flask filled with 50mL of LB medium, and cultivate for 8 hours at a rotary shaker with a rotational speed of 200r / min and a cultivation temperature of 37°C. Fermentation culture: Inoculate the cultivated seed culture solution into a 500mL Erlenmeyer flask containing 100mL fermentation medium at an inoculum size of 4% (v / v) for cultivation. The initial cultivation temperature is 30°C, and the shaker speed is 200r / min , when the cells were cultured to OD 600 When it is 0.6, after adding IPTG, quickly transfer to a shaker at different temperatures, and continue to induce for 90h. Add 100 μg / mL ampicillin to each medium before use.

[0046] After fermentation, the fermentation supernatant was taken for SDS-PAGE electrophoresis ( figure 2 ), and measure the enzyme activity of the fermentation supernatant. The activity of cyclo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Belonging to the technical field of genetic engineering, the invention discloses a repetitive extragenic palindromic sequence for improving exogenous gene mRNA and application thereof. The repetitiveextragenic palindromic sequence and a spacer region sequence thereof are shown as SEQ ID NO, 1, the signal peptide can improve the stability of mRNA by changing the secondary structure, so that the extracellular enzyme activity of the target protein cyclodextrin glycosyltransferase can be improved by 14%, the recombinant escherichia coli modified by the signal peptide has strengthened extracellular protein production capacity, therefore the repetitive extragenic palindromic sequence can be used for escherichia coli to express exogenous protein.

Description

technical field [0001] The invention relates to a method for improving the repetitive palindromic sequence of exogenous gene mRNA and its application, which belongs to the technical field of genetic engineering. Background technique [0002] Cyclodextrin glucosyltransferase (CGTase for short) has a wide range of applications and is mainly used to catalyze the production of cyclodextrin. Cyclodextrin glucosyltransferase converts starch to cyclodextrins by cyclization. In addition, cyclodextrin glucosyltransferase can be used to catalyze the transfer of one or more sugar groups to carbohydrates to improve their properties (such as increased solubility, stability, reduced cytotoxicity, bitterness, etc.). Cyclodextrin glucosyltransferases are often heterologously expressed in E. coli to enhance their production. However, it is very limited to increase the yield of cyclodextrin glucosyltransferase through simple heterologous expression, and it is often necessary to further enha...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/70C12N15/11C12N15/54C12N9/10C12N1/21C12R1/19
CPCC12N9/1074C12N15/70C12N2840/105
Inventor 刘龙堵国成陈坚李江华邓琛
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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