Novel endoribonuclease
A ribonucleic acid and endonuclease technology, applied in hydrolase, cells modified by introducing foreign genetic material, recombinant DNA technology, etc.
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Embodiment 1
[0067] Example 1: Isolate DR0662 from Micrococcus radioresistant R1, isolate Mb2014c homologue from Mycobacterium bovis BCG and construct expression plasmid
[0068] The amino acid and nucleotide sequences of the polypeptides encoded by the DR0662 gene from Micrococcus radioduranus R1 and the Mb2014c homologue gene from Mycobacterium bovis BCG and their nucleotide sequences can be obtained from the NCBI database (accession numbers NP_294385 and NC_001263, and NP_855664 and NC_002945 ). Primer DR0662-F (SEQ ID NO: 5) and primer DR0062-R (SEQ ID NO: 6) were synthesized according to the relevant information of DR0662 nucleotide sequence and used for PCR reaction to amplify the DNA region encoding the entire polypeptide.
[0069]Micrococcus radiodurans R1 genomic DNA can be obtained from ATCC (ATCC No. 13939D). PCR was performed using Pyrobest DNA polymerase (Takara Bio) and 50 ng of genomic DNA from Micrococcus radiodurans R1 and primers DR0662-F and DR0662-R to obtain a 368-bp ...
Embodiment 2
[0071] Example 2: Preparation of the DR0662 polypeptide from Micrococcus radiodurans R1 and the Mb2014c homologue polypeptide from Mycobacterium bovis
[0072] Example 1 transforms Escherichia coli BL21 (DE3) (Novagen) with the obtained expression vector pET-DR0662 or pET-Mb2014cHlg, obtains Escherichia coli, pET-DR0662 / BL21 (DE3) and pET-Mb2014cHlg / BL21 (DE3) for expression ). pET-DR0662 / BL21(DE3) or pET-Mb2014cHlg / BL21(DEB) was cultured in 5 ml LB medium containing 100 ug / ml ampicillin at 37°C. When OD600nm reached 0.6, IPTG (Takara Bio) was added to a final concentration of 1 mM to induce expression of the polypeptide. The culture was stopped two hours after the initiation of the induction, and the cells were collected by centrifugation. Suspend the cells in 300 μl lysis buffer (50 mM NaH 2 PO 4 , 300mM NaCl, 10mM imidazole, pH 8.0), it was disrupted by a sonicator (Handy sonic, Tomy). 20 μl of Ni-NTA agarose (Qiagen) was added, the supernatant was collected by centrif...
Embodiment 3
[0073] Embodiment 3: Use oligoribonucleotide as substrate to identify the nucleotide sequence specificity of DR0662 polypeptide and Mb2014c homologue polypeptide
[0074] Oligoribonucleotides were synthesized and cleavage assays were performed to study the nucleotide sequence specificity of the ribonuclease activity of the DR0662 polypeptide obtained in Example 2 and the Mb2014c homologue polypeptide.
[0075] Seven oligoribonucleotides in SEQ ID NO: 8-14 were synthesized as substrates. Incubate at 37° C. for 30 minutes with 5 μl of a reaction mixture consisting of 10 μM of one of the above oligoribonucleotides, 2.5 ng / μl of the DR0662 polypeptide or Mb2014c homologue polypeptide obtained in Example 2, and 10 mM Tris-HCl (pH 7.5). The reaction product was electrophoresed with 20% denaturing acrylamide gel (20% acrylamide, 7M urea, 0.5×TBE buffer). After staining with SYBR GREEN II (Takara Bio), the fluorescence image was analyzed with a fluorescence image analyzer FMBIO II Mu...
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