A kind of recombinant nuclease and preparation method thereof

A technology for recombining nucleic acid and enzyme genes, applied in the biological field, can solve the problem of product inactivity, achieve high expression, simple expression scheme, and improve quality

Active Publication Date: 2021-06-18
HANGZHOU JUNFENG BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

but the product is inactive

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  • A kind of recombinant nuclease and preparation method thereof
  • A kind of recombinant nuclease and preparation method thereof
  • A kind of recombinant nuclease and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Obtaining of recombinant nuclease gene

[0068] According to the Serratia marcescens (S.marcescens) nuclease gene sequence reported in the literature (GenBank: M19495.1), the gene expression sequence was optimized. The optimized sequence is shown in Sequence Table 1, and 20 complementary oligonucleotides were synthesized. According to the conventional method of molecular cloning, first treat with T4 phage polynucleotide kinase at 37°C for 30 minutes, mix the phosphorylated oligonucleotide fragments in equimolar ratio, denature at 94°C for 5 minutes, immediately anneal at 65°C for 10 minutes, then add T4 ligase , ligated overnight at 16°C to obtain target gene template fragments. Take 4 sterile microcentrifuge tubes and add:

[0069]

[0070] The above mixture was shaken gently to mix well, then briefly centrifuged, and then placed in a 14°C water bath for incubation and connection overnight (12-16h). Take 5 μl of the ligation product and add it to 50 μl E...

Embodiment 2

[0071] Example 2 Expression of N-terminal fusion tag recombinant nuclease gene

[0072] Design a pair of primers. Primers 1 and 2 are shown in Sequence Listing Sequence 2 and Sequence 3 respectively. The primer at the 5' end of the gene has an Nde I restriction site, and the primer at the 3' end has a BamH I restriction site. In 0.2ml PCR Prepare a 25μl reaction system in a microcentrifuge tube:

[0073]

[0074] Pre-denature at 94°C for 5 minutes, set 94°C for 1min, 55°C for 1min, 72°C for 2min, a total of 30 cycles, and finally 72°C for 10min. After PCR, 10 μl of the product was taken for agarose gel electrophoresis, and the fragment size was consistent with the designed size of 777 bp, see sequence 4 in the sequence listing, and sequence 5 for the encoded amino acid sequence.

[0075] The pET-22a plasmid was digested with Nde I and BamH I to recover the large fragment, and ligated with the nuclease gene fragment of PCR. The ratio of the gene fragment to the large vector...

Embodiment 3

[0078] Example 3 Expression of the recombinant nuclease gene with a fusion tag at the C-terminus

[0079] Design a pair of primers, primers 3 and 4 are shown in the sequence table sequence 6 and sequence 7 respectively, the gene 5' end primer has a Nde I restriction site, and the 3' end primer has a BamH I restriction site, in 0.2ml PCR Prepare a 25μl reaction system in a microcentrifuge tube:

[0080]

[0081] Pre-denature at 94°C for 5 minutes, set 94°C for 1min, 56°C for 1min, 72°C for 2min, a total of 30 cycles, and finally 72°C for 10min. After the PCR, 10 μl of the product was taken for agarose gel electrophoresis, and the fragment size was consistent with the designed size of 784 bp, see sequence 8 in the sequence listing, and sequence 9 for the encoded amino acid sequence.

[0082] The pET-11b plasmid was digested with Nde I and BamH I to recover the large fragment, and ligated with the nuclease gene fragment of PCR. The ratio of the gene fragment to the large vect...

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Abstract

The invention relates to the field of biotechnology, in particular to a nuclease and a preparation method thereof. The recombinant nuclease of the present invention is composed of Serratia marcescens extracellular nuclease and fusion tag peptide. The present invention also provides a method for preparing a recombinant nuclease, including optimizing the gene sequence, linking the coding sequence of the fusion tag peptide with the nuclease gene, cloning, transforming and screening high-expression strains, culturing and performing separation and purification to obtain a high-purity recombinant Nuclease. The recombinant nuclease and the preparation method thereof of the present invention have simple expression scheme, high expression amount up to 30%, strong purification process specificity and high yield. The recombinant nuclease of the invention is applied in the process of pharmaceutical products, and can be easily removed from the product by affinity chromatography in the process of separation and purification of the product, which helps to improve product quality.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nuclease and a preparation method thereof. Background technique [0002] When biotechnology products are highly expressed, the host cells or bacteria also proliferate in large quantities. This process is often accompanied by the amplification of a large number of host or bacteria nucleic acids. In the subsequent isolation process, a large number of nucleic acids are released. Purification increases the difficulty, and at the same time, it is difficult to remove some residual host DNA combined with the product. The DNA of the host cell entering the human body together with the drug will produce unpredictable side effects. The residual DNA may cause the insertion mutation of the DNA of the body, resulting in the inactivation of the tumor suppressor gene, the activation of the oncogene, etc., which may cause the drug user to cause cancer. Therefore, the host cell DNA residue is one o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12N15/70C12R1/43
CPCC12N9/22C12Y301/30002
Inventor 刘国安徐辉方芳
Owner HANGZHOU JUNFENG BIOENG
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