Recombinant nuclease and preparation method thereof

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

Active Publication Date: 2014-10-15
HANGZHOU JUNFENG BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Recombinant nuclease and preparation method thereof

Examples

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Embodiment 1

[0066] Example 1 Obtaining of Recombinant Nuclease Gene

[0067] According to the nuclease gene sequence of S. marcescens (S. marcescens) 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 bacteriophage polynucleotide kinase at 37°C for 30min. The phosphorylated oligonucleotide fragments are mixed in equal moles, denatured at 94°C for 5min, immediately annealed at 65°C for 10min, and then added T4 ligase Ligated overnight at 16°C to obtain the target gene template fragment. Take 4 sterilized microcentrifuge tubes and add:

[0068]

[0069] The above mixture was shaken gently and then centrifuged briefly, and then placed in a water bath at 14°C for incubation and connection overnight (12-16h). Take 5μl of the ligation product and add it to 50μl of E. coli ...

Embodiment 2

[0070] Example 2 Expression of N-terminal fusion tag recombinant nuclease gene

[0071] Design a pair of primers, primers 1 and 2, see sequence 2 and sequence 3 in the sequence table respectively. The 5'end primer of the gene has an Nde I restriction site, and the 3'end primer has a BamH I restriction site. In 0.2ml PCR Prepare 25μl reaction system in a microcentrifuge tube:

[0072]

[0073] Pre-denaturation at 94°C for 5 minutes, setting 94°C for 1 minute, 55°C for 1 minute, 72°C for 2 minutes, 30 cycles in total, and finally 72°C for 10 minutes. After PCR, 10μl of the product was taken and subjected to agarose gel electrophoresis. The size of the fragment was consistent with the designed size of 777bp. See sequence 4 in the sequence listing, and the encoded amino acid sequence see sequence 5 in the sequence listing.

[0074] The pET-22a plasmid was digested with Nde I and BamH I to recover large fragments, and ligated with PCR nuclease gene fragments. The ratio of 20μL reaction s...

Embodiment 3

[0077] Example 3 Expression of C-terminal fusion tag recombinant nuclease gene

[0078] Design a pair of primers, primers 3 and 4 are shown in sequence 6 and sequence 7, respectively. The 5'end primer of the gene has an Nde I restriction site, and the 3'end primer has a BamH I restriction site. In 0.2ml PCR Prepare 25μl reaction system in a microcentrifuge tube:

[0079]

[0080] Pre-denaturation at 94°C for 5 minutes, setting 94°C for 1 minute, 56°C for 1 minute, 72°C for 2 minutes, 30 cycles in total, and finally 72°C for 10 minutes. After PCR, 10μl of the product was taken and subjected to agarose gel electrophoresis. The size of the fragment was consistent with the designed size of 784bp. See sequence 8 in the sequence listing, and the encoded amino acid sequence see sequence 9 in the sequence listing.

[0081] The pET-11b plasmid was digested with Nde I and BamH I to recover large fragments, and ligated with PCR nuclease gene fragments. The ratio of 20μL reaction system gene fr...

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Abstract

The invention relates to the technical field of biology and especially to a recombinant nuclease and a preparation method thereof. The recombinant nuclease is composed of Serratia marcescens extracellular nuclease and a fusion tag peptide fragment. The invention further provides the preparation method for the recombinant nuclease. The preparation method comprises the following steps: optimization of a gene sequence; connection of the coding sequence of the fusion tag peptide fragment and a nuclease gene, cloning and transformation; screening of high-expression bacterial strains and culture; and separation and purification so as to obtain the high purity recombinant nuclease. According to the recombinant nuclease and the preparation method thereof in the invention, an expression scheme is simple, an expression level is high, up to 30%, and a purifying technology has strong specificity and high yield. The recombinant nuclease is applied to technology for medicine products and can be easily removed from products by using affinity chromatographic technology during separation and purification of products, which is beneficial for improvement of 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 efficiently expressed, host cells or bacteria also proliferate in large quantities. This process is often accompanied by the amplification of a large number of host or bacterial nucleic acids. In the subsequent separation process, a large amount of nucleic acid release will give the target product separation. Purification increases the difficulty, and it is also difficult to remove part of the residual host DNA combined with the product. The DNA of the host cell enters the human body together with the drug will cause unpredictable side effects. Residual DNA may cause insertion mutations in the body's DNA, leading to inactivation of tumor suppressor genes, activation of oncogenes, etc., which may cause cancer in drug users. Therefore, host cells DNA residue is one of the important in...

Claims

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Application Information

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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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