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mhp non-specific nuclease and its coding gene and application

A non-specific, nuclease technology, applied in the field of Mhp non-specific nuclease and its encoding gene and application, can solve the problems of high price, large-scale application limitation, difficulty in renaturation, etc., achieve low ion concentration, reduce trapping and killing , enhance the effect of infection and pathogenesis

Active Publication Date: 2021-02-19
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recombinant protein has a signal peptide, some of which exist in the form of inclusion bodies, and it is difficult to refold, which makes it expensive and limits its large-scale application

Method used

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  • mhp non-specific nuclease and its coding gene and application
  • mhp non-specific nuclease and its coding gene and application
  • mhp non-specific nuclease and its coding gene and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The acquisition and point mutation of embodiment 1Mhp non-specific nuclease nucleotide sequence

[0041] Utilize the known Mycoplasma hyopneumoniae (Mhp) non-specific nuclease gene (gene accession number is AAZ53943.1) to design primers, Mhp597-F'(5'-ATGAAAATTAAAAAATTATTTTCTTTTT-3') and mhp597-R'(5'-TTAATTCTGACTGTTTTGGCCT- 3'), using the whole Mhp genome as a template, carry out PCR amplification, connect the obtained sequence to the cloning vector and send it to Meiji Biotechnology Co., Ltd. for sequencing. The sequencing results were translated into protein sequences using DNAStar software, and the TGA stop codon inside the gene fragment of Mhp non-specific nuclease was found. Mutation primers were designed. The list of mutation primers is shown in Table 1. Point mutations were carried out by overlapping extension PCR. The TGA inside the gene is mutated into TGG to obtain the nucleotide sequence of the Mhp non-specific nuclease. The result is shown in SEQ ID NO:1, and...

Embodiment 2

[0047] Embodiment 2 expresses the construction of Mhp non-specific nuclease engineering strain

[0048] 1. Amplification of Mhp non-specific nuclease gene

[0049] Design primers according to the modified Mhp non-specific nuclease gene sequence characteristics, the upstream primer Mhp597-F contains NcoI site Mhp597-F (5'-CATGCCATGGGCACCGGAC TCGGAGC TTATTT-3'), does not amplify the original signal peptide (1-18 Amino acid removal, the total length is 54bp), the downstream primer Mhp597-R contains the XhoI site Mhp597-R (5'-CCGCTCGAGATTCTGACTGTTTTGG CCT-3'), the Mhp non-specific nuclease gene (shown in SEQ ID NO: 1) is used as a template, and the PCR amplification. The PCR amplification system and conditions are the same as in Example 1.

[0050] 2. Cloning of Mhp non-specific nuclease gene

[0051] Use the ordinary agarose gel DNA recovery kit of Beijing Tiange Biochemical Technology Co., Ltd. to recover the PCR amplification product of step (1), and connect it to the pEASY-...

Embodiment 3

[0059] The enzymatic property research of embodiment 3Mhp non-specific nuclease

[0060] 1. Degradation of different nucleic acid substrates by Mhp non-specific nucleases

[0061] Use dsDNA (calf thymus DNA), ssDNA, plasmamid DNA, and RNA as substrates, add 1 μg of Mhp597 protein, and put 2+ , 100mM Mg 2+ In the buffer solution (Table 2), react for 0.25min, 1min, 2min, 3min, 4min, 5min respectively, and then run the reaction system on 1% agarose gel electrophoresis. The results show that 1 μg Mhp non-specific nuclease can completely degrade 1 μg double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA and RNA, and the degradation reaction time is extremely short, and the degradation reaction can be completed by mixing ( figure 2 A, figure 2 B, figure 2 C, figure 2 D).

[0062] Table 2Mhp non-specific nucleases degrade different nucleic acid substrates

[0063]

[0064] 2. Effect of protein concentration on Mhp non-specific nuclease activity

[0065] I...

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Abstract

The present invention relates to Mhp non-specific nuclease and its encoding gene and application. The amino acid sequence of the Mhp non-specific nuclease is shown in SEQ ID NO:2. The Mhp non-specific nuclease provided by the invention can degrade various nucleic acid substrates, such as single-stranded DNA, double-stranded DNA, plasmid and RNA, has high enzymatic activity, and high temperature resistance, and can be widely used in biopharmaceuticals and biological reagents Research and development, industrialization and other fields.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to Mhp non-specific nuclease and its coding gene and application. Background technique [0002] Nuclease is a type of enzyme that can degrade nucleic acid substrates and catalyze the hydrolysis of phosphodiester bonds. It can be divided into three categories: DNase, RNase and non-specific nuclease. Among them, non-specific nucleases are a class of highly active hydrolytic enzymes that can non-specifically degrade almost all forms of nucleic acids, including single-stranded, double-stranded, linear, circular and supercoiled forms of DNA and RNA, and can Sequence is not required. Nonspecific nucleases have many important functions, such as participating in DNA replication, recombination and repair, RNA splicing, maturation, degradation and interference, vegetative growth of cells, apoptosis, inflammatory response, vascular thrombosis and host defense, etc. Therefore, it has important ap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/22C12N15/70
Inventor 吴文学李鹏张云科
Owner CHINA AGRI UNIV
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