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A kind of dna for increasing the amount of foreign gene mRNA and its application

An exogenous gene and quantitative technology, applied in the field of DNA and its application, can solve the problem of limited production of cyclodextrin glucosyltransferase and achieve the effect of increasing expression

Active Publication Date: 2020-11-06
JIANGNAN UNIV
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  • 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

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  • A kind of dna for increasing the amount of foreign gene mRNA and its application
  • A kind of dna for increasing the amount of foreign gene mRNA and its application
  • A kind of dna for increasing the amount of foreign gene mRNA and its application

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 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...

Embodiment 4

[0047] Embodiment 4 real-time fluorescent quantitative PCR verification

[0048] Using the strain containing the plasmid CGT-P1-1-DB3 as a control, the fermentation supernatant of the 24h, 48h, and 72h of extraction and fermentation was centrifuged to remove the supernatant, and then the bacteria were ground under liquid nitrogen, and then according to the Takar company's RNA Extraction kit instructions for RNA extraction. After confirming that the RNA concentration meets the requirements, the reverse transcription kit from Takara Company was used to prepare cDNA. Use this as a template to use Takara's Premix Ex Taq TM II (Tli RNaseH Plus) kit for fluorescent quantitative PCR (qRT-PCR) analysis. The transcription levels of 16sRNA in the two strains were used as the internal reference, and the primers 16SrRNAF / 16SrRNA R were used for qRT-PCR determination; the target gene CGT was determined by qRT-PCR with primers RTCGT1 F / RTCGT1 R.

[0049] RTCGT1 F:CCTGATGGACGAGATTGA; ...

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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 DNA for increasing the amount of exogenous gene mRNA and its application, belonging 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 enhance its heterologous expression...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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