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Human Annexin V genetic optimization sequence and preparation method and application thereof

A technology for gene optimization and sequence optimization, applied in the field of bioengineering, can solve problems such as difficult recombination expression, and achieve the effects of reducing production costs, simple equipment and process, and easy large-scale production.

Active Publication Date: 2015-09-16
苏州四正柏生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention solves the problem of difficult recombinant expression of human Annexin V protein, greatly reduces the production cost of the apoptosis detection kit, and has a good market application prospect

Method used

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  • Human Annexin V genetic optimization sequence and preparation method and application thereof
  • Human Annexin V genetic optimization sequence and preparation method and application thereof
  • Human Annexin V genetic optimization sequence and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A kind of optimized method of human Annexin V gene:

[0032] Log in to GeneBank and search for the gene sequence encoding the mature protein of human Annexin V (sequence number: GenBank: AK312644.1). Its nucleotide length is 963bp. Since human Annexin V is a eukaryotic protein, follow-up research needs to be carried out in prokaryotes Therefore, the coding codons of the above-mentioned human Annexin V gene were compared with the preferred codons of E. coli. According to the degeneracy of the codons, on the basis of not changing the sequence of the amino acid composition, the known human Annexin V coding The gene sequence is modified, and some rare codons of E. coli are replaced with preferred codons of E. coli to improve the expression level of the target gene in E. coli, and the transgenic verification is carried out. The finally obtained human Annexin V gene optimized sequence is shown as SEQ ID NO: 1.

[0033] Add the NdeI restriction site to the 5′ end of the human...

Embodiment 2

[0035] 2.1 Obtaining expression strains

[0036] The prokaryotic expression plasmid pET23a-ANXV obtained in Example 1 was transformed into Escherichia coli BL21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) by the heat shock method, and ampicillin was added to the LB plate medium to 100 mg / L was screened, and a single colony was picked for sequencing identification.

[0037] 2.2 Induced expression of human Annexin V gene optimized sequence in Escherichia coli

[0038] Pick a single colony containing the recombinant positive plasmid, inoculate it into LB liquid medium containing 100mg / L ampicillin, culture overnight at 37°C and 220rpm with shaking, and inoculate the overnight cultured bacterial solution to 100mg / L ampicillin fresh LB liquid medium, 37 ° C, 220rpm shaking culture to the bacterial concentration OD 600 About 0.6, take out 2mL of the bacterial liquid as the uninduced control, immediately add the inducer IPTG to the working concentration of 1...

Embodiment 3

[0043] Human Annexin V FITC labeling and apoptosis detection

[0044] 3.1 Human Annexin V FITC labeling

[0045] Take 5 mg of human Annexin V recombinant protein, and use a dialysis bag with a cut-off molecular weight of 12-14KDa for overnight dialysis (buffer 0.1M Na 2 CO 3 -NaHCO 3 , pH9.5) dialyzed, during which the buffer was changed 3 times; 4°C, 12000rpm, centrifuged for 15min; the supernatant was taken to measure the concentration. Dissolve FITC in DMSO to a final concentration of 5mg / mL; add FITC to the protein according to the ratio of 9uL FITC per mg of human Annexin V recombinant protein, mix with a vortex mixer, wrap with tinfoil, and rotate in a hybridization oven at room temperature in the dark Reaction for 2 hours; equilibrate Sephadex G25 chromatography column with 5 times column bed volume deionized water; 5 times column bed volume Annexin V-FITC storage buffer (50mM Tris, 150mM NaCl, 0.05%NaN 3 , pH8.0) to equilibrate the Sephadex G25 chromatography colum...

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Abstract

The invention optimizes the nucleotide sequence of the human Annexin V gene, obtains the optimized gene of the human Annexin V by utilization of the whole-genome synthetic technology, builds an artificially synthesized prokaryotic expression vector of the human Annexin V genetic optimization sequence , converts the prokaryotic expression vector of the above human Annexin V genetic optimization sequence into the escherichia coli for efficient inducible expression, extracts and purifies the expression protein, and marks the purified human Annexin V protein FITC in the apoptosis detection test for conducting the biological active validation. The expression quantity of the artificially synthesized human Annexin V genetic optimization sequence in the escherichia coli reaches 200mg / 1L bacterium solution, and the biological activity achieves the effects equal to similar products of the international famous brands. The human Annexin V protein produced according to the human Annexin V genetic optimization sequence is simple in process, high in expression quantity, good in purity, low in cost and good in biological function, and capable of producing the apoptosis detection products, greatly reducing the needs for imported raw materials and having a broad market application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an optimized human Annexin V gene sequence and a production method and application thereof. Background technique [0002] Human annexin V (Annexin V) is a calcium ion-dependent phospholipid-binding protein—one of the members of the Annexin family. A total of 320 amino acids, with a molecular weight of about 35.8 KDa, exists in most eukaryotic cells and is expressed in various tissue cells (such as cardiomyocytes, vascular endothelium, skeletal muscle, and liver cells, etc.). in ca 2+ When present, it has a high affinity for acidic phospholipid molecules, and has a variety of physiological functions in the body. It participates in membrane transport in cells and a series of calmodulin-dependent activities on the membrane surface, including membrane fusion in exocytosis, signaling Conduction and formation of calcium ion channels, anticoagulation, anti-inflammatory respon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K14/47G01N15/14
Inventor 杨艳梅
Owner 苏州四正柏生物科技有限公司
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