Novel endoribonuclease
A technology of endoribonucleic acid and enzymatic activity, applied in the field of genetic engineering
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Embodiment 1
[0068] Example 1: Isolate PH1182 from Pyrococcus Horikoshii ATCC700860, and construct an expression plasmid nucleotide sequence. Primer PH1182-F (SEQ ID NO: 3) and primer PH1182-R (SEQ ID NO: 4) for PCR amplification of the DNA region encoding the complete polypeptide were synthesized based on the nucleotide sequence information of PH1182.
[0069] Pyrococcus horikoshii ATCC700860 genomic DNA was obtained from ATCC (ATCC No. 700860D). PCR was performed with Pyrobest DNA polymerase (Takara Bio) and 50 ng of genomic DNA from Pyrococcus horikoshii ATCC700860 and primers PH1182-F and PH1182-R to obtain a 437-bp amplified DNA fragment. This amplified fragment was digested with restriction enzymes NdeI and XhoI, and subjected to agarose gel electrophoresis, and a 416-bp DNA fragment was recovered from the gel.
[0070] Expression vectors were constructed using pCold TF (Takara Bio). In order to introduce a stop codon after gene cloning following the XhoI site at the 3' end of the...
Embodiment 2
[0072] Example 2: Preparation of PH1182 polypeptide derived from Pyrococcus Horikoshii ATCC700860
[0073] Escherichia coli BL21(DE3) (Novagen) was transformed with the expression vector pCold TF-PH1182 obtained in Example 1 to obtain Escherichia coli pCold TF-PH1182 / BL21(DE3) for expression. Escherichia coli cells were cultured at 37°C in 5 ml of LB medium containing 100 µg / ml ampicillin. When OD600nm reached 0.5, incubate at 15°C for 30 minutes, add IPTG (Takara Bio) at a final concentration of 1 mM to induce polypeptide expression, and continue culturing at 15°C for 24 hours. The culture was terminated after 24 hours, and the cells were collected by centrifugation. Cells were suspended in 300 μl lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0), and disrupt the cells with a sonicator (Handy sonic, Tomy). To the supernatant collected by centrifugation was added 20 μl of Ni-NTA agarose (Qiagen), and the mixture was allowed to stand at 4° C. for 30 min...
Embodiment 3
[0074] Example 3: Using oligoribonucleotides as a substrate to identify the nucleotide sequence specificity of the PH1182 polypeptide
[0075] Oligoribonucleotides were synthesized and cleavage assays were performed to investigate the nucleotide sequence specificity of the ribonuclease activity of the PH1182 polypeptide obtained from Example 2.
[0076] Eleven oligoribonucleotides of SEQ ID NOS: 7-17 were synthesized as substrates. A 5-µl reaction mixture consisting of 10 µM of one of the oligoribonucleotides, 5 ng / µl of the PH1182 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, 0.5×TBE buffer). After staining with SYBR GREEN II (Takara Bio), the fluorescence image was analyzed with a fluorescence image analyzer FMBIO II Multiview (Takara Bio). The cleavage pattern of each oligoribonucleotide is shown in Table 1.
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