A cold-adapting ribonuclease r and its encoding gene and application

A ribonuclease and cold ribonucleic acid technology, which is applied to cold-adapting ribonuclease R and its encoding gene and application field, can solve problems such as poor stability and the like

Active Publication Date: 2022-08-02
HARBIN INST OF TECH AT WEIHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The ribonuclease R extracted from animals and plants is easily restricted by season, climate and region, and its stability is poor, so it is difficult to be directly applied to related fields such as industrial production

Method used

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  • A cold-adapting ribonuclease r and its encoding gene and application
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  • A cold-adapting ribonuclease r and its encoding gene and application

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Experimental program
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Effect test

Embodiment 1

[0018] Cloning and sequencing analysis of ribonuclease R gene.

[0019] Antarctic Microbes Psychrobacter sp. ANT206 was activated in 2216E liquid medium and cultured to the middle and late logarithmic growth phase (about 4 days), and the total gene DNA of the strain was extracted by combining CTAB method and phenol-chloroform extraction method. PCR was performed using degenerate primers using the extracted total DNA as a template.

[0020] Upstream primer: 5'-ATGANGGAATGATCRACGCCT-3'

[0021] Downstream primer: 5'-ANGTTTGGATCATTNACTCAT-3'

[0022] The amplification conditions were: denaturation at 94 °C for 1 min, annealing at 54 °C for 1 min, and extension at 72 °C for 90 s, with 30 cycles. Then, the band containing the target gene cryogenic ribonuclease R was detected by agarose gel electrophoresis and sequenced. After analyzing the sequencing results, a gene with a complete reading frame sequence of 2 313 bp in full length was obtained. The nucleotide sequence is shown ...

Embodiment 2

[0024] Expression and purification of ribonuclease R gene

[0025] The primers containing the restriction enzyme cleavage site were redesigned according to the determined full-length sequence of cold-adapted ribonuclease R.

[0026] Upstream primer: 5'-ACT GGATCC ATGTCAAACCAAGATC-3’

[0027] Downstream primer: 5'-TAC CTCGAG CGCTCTTTTTACTACT -3'

[0028] The lines are respectively Bam HI, Xho I restriction site.

[0029] The cold-adapting ribonuclease R gene and pET-28a(+) double-enzyme digested gel-recovered products were ligated by T4 ligase in proportion to construct a recombinant expression vector. Transform the recombinant expression vector into competent cells E. coli In BL21, positive clones were screened and verified by enzyme digestion.

[0030] The recombinant strains obtained by screening were induced to express by IPTG. The recombinant bacteria were inoculated into LB medium and cultured to OD at 32-40 °C 600 IPTG was added to the medium to a final c...

Embodiment 3

[0032] Study on the Enzymatic Properties of Recombinant Cold-adaptive Ribonuclease R

[0033] (1) Determination of the optimum reaction temperature: Under the conditions of different temperatures (0 ~ 50 ℃) and pH 6.0, the enzyme activity of cold-adaptive RNase R was determined, and the highest enzyme activity was set as 100%, and the enzyme activity at other temperatures was determined. The ratio of the enzyme activity to the highest enzyme activity is the relative enzyme activity of the enzyme solution to be tested at this temperature. The result is as figure 2 The results show that the optimal temperature of the enzyme is between 28 and 32 °C, and the enzyme activity is maintained at more than 85% of the highest enzyme activity at 20 to 35 °C. When the temperature increased, the enzyme activity gradually decreased, and when the temperature reached above 50 °C, the enzyme activity was basically lost.

[0034] (2) The effect of different concentrations of NaCl on enzyme ac...

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Abstract

The present invention discloses a cold-adapting ribonuclease R and its encoding gene and application. The present invention is firstly obtained from Antarctic sea ice microorganism psychrophilic bacillus ( Psychrobacter sp.) cloned a novel cold-adapting ribonuclease R-encoding gene, and the amino acid residue sequence is shown in SEQ ID No.2. Sequence analysis showed that the protein sequence encoded by the gene contained a total of 770 amino acid residues, belonging to the RNR superfamily, and the highest homology with the published protein sequences in the current database was 90.07%. Its expression mode is to use plasmid pET-28(+) to construct a recombinant expression vector containing ribonuclease R gene, and transfer the constructed recombinant expression vector into Escherichia coli host cells ( Escherichia coli ), which was induced to express the RNase R gene. The optimal catalytic temperature of the expression product ribonuclease R of the present invention is about 30° C., and more than 50% of the highest enzymatic activity is maintained between 15 and 40° C.; and more than 85% of the highest enzymatic activity is maintained in the presence of 0-3 M NaCl. ,with broadly application foreground. The expression product of the invention has outstanding cold adaptability, good stability and certain salt tolerance, which opens up potential applications in food, medicine and molecular biology related fields.

Description

technical field [0001] The invention belongs to the related fields of enzyme engineering, food, medicine and the like, and in particular relates to a cold-adapting ribonuclease R and its encoding gene and application. Background technique [0002] Ribonuclease (RNase) is an important nucleic acid hydrolase in organisms. Its function is to hydrolyze phosphodiester bonds between nucleic acid residues to generate mononucleotides or oligonucleotides, which play a role in RNA metabolism. important role. Because of its special biological activity and pharmacological function, the RNase family has high application value in the fields of food, medicine and molecular biology research. Ribonuclease R is a member of the ribonuclease family and mainly acts on the 3'-5' phosphodiester bond in nucleic acids. Ribonuclease R is involved in controlling the type and quantity distribution of RNA in cells, and can also modify mRNA and degrade different types of RNA (including tRNA and rRNA). ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/22C12N15/70C12N1/21C12R1/19
CPCC12N9/22C12N15/70
Inventor 侯艳华王亚彤王全富王一帆
Owner HARBIN INST OF TECH AT WEIHAI
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