Protein having ribonuclease activity as well as preparation method and application of protein

A ribonuclease and protein technology, applied in the biological field, can solve problems such as elevation and infection

Inactive Publication Date: 2015-05-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the previous study of the applicant, it was found that the infection of Nicotiana benthamiana by the beet necrotic yellow vein virus (BNYVV) containing RNA4 components could induce a sharp increase of the mRNA encoding PR-10 protein (NbPR-10) of Nicotiana benthamiana (Wu et al ., 2014), using genetic engineering technology to establish a method for preparing NbPR-10 protein, it was proved that it has the activity of cutting single-stranded and double-stranded RNA in vitro, and PR-10, which has the activity of cutting double-stranded RNA in vitro, has not been reported yet.

Method used

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  • Protein having ribonuclease activity as well as preparation method and application of protein
  • Protein having ribonuclease activity as well as preparation method and application of protein
  • Protein having ribonuclease activity as well as preparation method and application of protein

Examples

Experimental program
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Embodiment 1

[0079] Example 1, protein NbPR-10 prokaryotic expression, purification and detection

[0080] 1. Construction of NbPR-10 gene expression plasmid

[0081] Replace the DNA between the Nco I recognition sequence and the Xho I recognition sequence of pGEX-KG with a DNA molecule whose nucleotide sequence is the 1-483 position of SEQID No.1, and keep the other sequences of pGEX-KG unchanged to obtain NbPR- 10 gene expression vector, its name is pGEX-NbPR-10. pGEX-NbPR-10 expresses the protein NbPR-10 shown in SEQ ID No.2. In pGEX-NbPR-10, the insertion direction of the NbPR-10 gene is consistent with the GST reading frame.

[0082]Replace the DNA between the Bam HI recognition sequence and the Hind III recognition sequence of pGEX-KG with the DNA molecule whose nucleotide sequence is the 1-720th position of SEQ ID No.3, and keep the other sequences of pGEX-KG unchanged The GFP gene expression vector is named pGEX-GFP. pGEX-GFP expresses the protein GFP shown in SEQ ID No.4. In ...

Embodiment 2

[0092] Embodiment 2, the detection of recombinant NbPR-10 protein ribonuclease activity

[0093] 1. Recombinant NbPR-10 protein cleaves total plant RNA in vitro

[0094] The total RNA (single-stranded RNA) of Nicotiana benthamiana was extracted by Trizol method. The GST-NbPR-10 sample and GST-GFP sample obtained through protein dialysis enrichment in Example 1 were adjusted to the protein content of GST-NbPR-10 and GST-GFP at 20°C with protein dissolution buffer to 2 mg / mL, GST-NbPR-10 solution and GST-GFP solution were obtained.

[0095] 1.1 Tobacco benthamiana total RNA GST-NbPR-10 treatment (referred to as benthamiana tobacco GST-Nb PR-10 treatment)

[0096] Take the above 3 μg total RNA of Tobacco benthamiana, add the above GST-NbPR-10 solution to obtain a 15 μL reaction system, make the protein content in the reaction system 6 μg, and add 6 × Loading Buffer to terminate the reaction, and then carry out agarose gel electrophoresis detection.

[0097] 1.2 Tobacco bentha...

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Abstract

The invention discloses a protein having ribonuclease activity as well as a preparation method and an application of the protein. The invention demonstrates that a plant PR-10 protein is a nuclease having activities of in-vitro-cleavage single-stranded RNA and double-stranded RNA and the following uses of the plant PR-10 protein belong to the scope of protection of the invention: C1, application of the protein as the ribonuclease or in preparation of ribonuclease products; and C2, application of protein-related biological materials in preparation of the ribonuclease products. The preparation method comprises the following steps: (1) preparing recombinant biological cells for expressing the protein; and (2) expressing the recombinant biological cells to obtain the protein. Experimental results show that the preparation method disclosed by the invention has strong practicability.

Description

technical field [0001] The invention relates to a protein with ribonuclease activity and its preparation method and application in the field of biotechnology. Background technique [0002] The full name of ribonuclease (Ribonuclease, RNase) is polyglycoside-α-oligonucleotide transferase, which is a class of nucleolytic enzymes widely present in animals and plants. It plays an important role in regulating RNA metabolism, post-transcriptional gene expression regulation and resisting virus infection (Court DL.et al., 2013). There are many types of nucleases that have been reported. For example, RNase A, which is commonly used in research, can hydrolyze RNA and can be used to digest RNA in DNA products; RNase L, which relies on the 2-5A system, can specifically degrade single-stranded RNA (Zhou A et al., 1993), RNaseIII family enzymes such as RNaseIII from Escherichia coli, Rnt1p and PacI from yeast, and Dicer from plants have ribonuclease activity that specifically cuts double...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/55C12N15/70C12N1/21
CPCC12N9/22
Inventor 韩成贵武文琦王颖姜宁范慧艳李大伟于嘉林张超王献兵张宗英张永亮
Owner CHINA AGRI UNIV
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