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Mhp non-specific nuclease and coding gene and application thereof

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

Active Publication Date: 2019-01-18
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 coding gene and application thereof
  • Mhp non-specific nuclease and coding gene and application thereof
  • Mhp non-specific nuclease and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Acquisition and point mutation of Mhp non-specific nuclease nucleotide sequence

[0041] Primers were designed using the known Mycoplasma hyopneumoniae (Mhp) nonspecific nuclease gene (gene accession number AAZ53943.1), Mhp597-F' (5'-ATGAAAATTAAAAAAATTATTTCTTTTT-3') and mhp597-R' (5'-TTAATTCTGACTGTTTTGGCCT- 3'), using the Mhp whole 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. Use DNAStar software to translate the sequencing results into protein sequences, find the TGA stop codon inside the gene fragment of Mhp non-specific nuclease, and design mutation primers. The TGA inside the gene is mutated into TGG, that is, the nucleotide sequence of the Mhp non-specific nuclease is obtained. The result is shown in SEQ ID NO: 1, and the corresponding amino acid sequence is shown in SEQ ID NO: 2.

[0042] PCR amplification system and conditions are as f...

Embodiment 2

[0047] The construction of embodiment 2 expressing Mhp non-specific nuclease engineering strain

[0048] 1. Amplification of Mhp nonspecific nuclease gene

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

[0050] 2. Cloning of Mhp nonspecific nuclease gene

[0051] Step (1) PCR amplification product was recovered by using the ordinary agarose gel DNA recovery kit of Beijing Tiangen Biochemical Technology Co., Ltd., and connec...

Embodiment 3

[0059] Example 3 Study on the enzymatic properties of Mhp non-specific nuclease

[0060] 1. Degradation of different nucleic acid substrates by Mhp nonspecific nucleases

[0061] Using dsDNA (calf thymus DNA), ssDNA, plasma DNA, and RNA as substrates, 1 μg Mhp597 protein was added, and 10mM Ca 2+ , 100mM Mg 2+ In the buffer (Table 2), react for 0.25min, 1min, 2min, 3min, 4min, 5min respectively, and then run the reaction system by 1% agarose gel electrophoresis. The results showed that 1 μg Mhp nonspecific nuclease could completely degrade 1 μg double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA and RNA, and the degradation reaction time was very short, and the degradation reaction could be completed by mixing ( figure 2 A, figure 2 B, figure 2 C, figure 2 D).

[0062] Table 2 Degradation of different nucleic acid substrates by Mhp nonspecific nucleases

[0063]

[0064] 2. The effect of protein concentration on the activity of Mhp non-specific nu...

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Abstract

The present invention relates to Mhp non-specific nuclease and coding gene and application thereof. 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 temperatureresistance, and can be widely used in the fields of biopharmaceutical, biological reagent research and development, industrialization and the like.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to Mhp non-specific nuclease and its encoding gene and application. Background technique [0002] Nucleases are a class of enzymes that can degrade nucleic acid substrates and catalyze the hydrolysis of phosphodiester bonds. Among them, non-specific nucleases are a class of highly active hydrolases 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. Sequence is not required. Nonspecific nucleases have many important functions, for example, involved in DNA replication, recombination and repair, RNA splicing, maturation, degradation and interference, cell vegetative growth, apoptosis, inflammatory response, vascular thrombosis and host defense and other processes, Therefore, it has important application value in the diagnosis and treatment of diseases such as cance...

Claims

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Application Information

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Patent Type & Authority Applications(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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