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: Isolation of NE1181 from Nitrosomonas Europe ATCC19718 to construct an expression plasmid
[0068] The amino acid sequence and its nucleotide sequence of the polypeptide encoded by the NE1181 gene from N. europeanum ATCC19718 can be obtained from the NCBI database (accession numbers NP_841237 and NC_004757). Primer NE1181-F (SEQ ID NO: 3) and primer NE1181-R (SEQ ID NO: 4) were synthesized according to the relevant information of NE1181 nucleotide sequence and used for PCR reaction to amplify the DNA region encoding the entire polypeptide.
[0069] Genomic DNA of N. europeanum ACTT19718 was obtained from ATCC (ATCC No. 19718D).
[0070] PCR was performed using Pyrobest DNA polymerase (Takara Bio) and 50 ng of genomic DNA from N. europeanum ATCC19718 and primers NE1181-F and NE1181-R to obtain a 362-bp amplified DNA fragment. The amplified fragment was hydrolyzed with restriction endonucleases NdeI and XhoI, and subjected to agarose gel electrophoresis, and a...
Embodiment 2
[0072] Example 2: Preparation of NE1181 polypeptide from N. europeanum ACTT19718
[0073] Example 1 The obtained expression vector pET-NE1181 was used to transform Escherichia coli BL21(DE3) (Novagen) to obtain Escherichia coli, pET-NE1181 / BL21(DE3) for expression. pET-NE1181 / 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 centrifugation, and the mixture was left at 4°C for 30 minutes. with 100 μl of wash buffer (50 mM NaH 2 PO 4 , 300mM NaCl,...
Embodiment 3
[0074] Embodiment 3: Use oligoribonucleotide as substrate to identify the nucleotide sequence specificity of NE1181 polypeptide
[0075] Oligoribonucleotides were synthesized and cleavage assays were performed to study the nucleotide sequence specificity of the ribonuclease activity of the NE1181 polypeptide obtained in Example 2.
[0076] Oligoribonucleotides in SEQ ID NO: 5-14 were synthesized as substrates. A 5 μl reaction mixture consisting of 10 μM of one oligoribonucleotide, 2.5 ng / μl of the NE1181 polypeptide obtained in Example 2, and 10 mM Tris-HCl (pH 7.5) was incubated at 37° C. for 30 minutes. The reaction product was electrophoresed on a 20% denaturing acrylamide gel (20% acrylamide, 7M urea, 0.5x TBE buffer). After staining with SYBR GREEN II (Takara Bio), the fluorescence image was analyzed with a fluorescence image analyzer FMBIO IIMultiview (Takara Bio). The cleavage mode of each oligoribonucleotide is shown in Table 1.
[0077] The cleavage patterns are as...
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