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Plectasin as well as gene and preparation method thereof

A kind of plectomycin and gene technology, applied in the field of bioengineering, can solve the problems of low yield of plectomycin, complicated purification steps, high difficulty and the like

Inactive Publication Date: 2013-10-30
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore the technical problem to be solved in the present invention is exactly: the expression level of existing Plectasin gene in fungus is low, it is difficult to carry out industrialization and the steps of purification are complicated, resulting in a relatively low yield of Plectasin The defect of plectasin provides a kind of preparation method of plectasin fusion protein and plectasin

Method used

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  • Plectasin as well as gene and preparation method thereof
  • Plectasin as well as gene and preparation method thereof
  • Plectasin as well as gene and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The design of embodiment 1.pletasin gene, the construction of synthesis and expression vector

[0056] 1.1 Design and synthesis of plectasin plectasin protein gene

[0057] The inventors designed the expression vector pYG330 of the plectasin plectasin protein, which can not only enable the plectasin plectasin fusion protein to be expressed in large quantities, but also does not affect the growth of the host Escherichia coli, and is convenient for subsequent separation and purification steps. The construction method of vector pYG330 comprises the following steps:

[0058] According to the published nucleotide sequence of plectasin in saprophytic ascomycetes (Per H.Mygind, Rikke L.Fischer, Kirk M.Schnorr et al, Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus[J].Nature , 437(13), October 2005: 975-980), and referring to the Escherichia coli codon preference usage table, designed and synthesized Mycelia expressing Plectasia and includ...

Embodiment 2

[0066] Example 2. Induced expression of fusion protein

[0067] 2.1 Plectasin fusion protein

[0068] The plectasin fusion protein of the present invention has a molecular weight of 25KD, which is co-expressed by a part of the protein expressed by the universal expression vector itself and the fragment inserted by the inventor, and terminates at the terminator TAG designed by the inventor, and the 4.7KD target protein is located at The end of the fusion protein, which is a part of the fusion protein as a whole, can be cut at the designed TEV restriction site to obtain the 4.7KD target protein. Among them, the 20.3KD large peptide is encoded by the nucleotide sequence of the expression vector backbone plasmid, and also contains the His-Tag tag designed by the inventor for easy separation and purification, and the amino acid sequence of the TEV enzyme recognition site.

[0069] 2.2 Induced expression of plectasin fusion protein

[0070] 1) Optimization of the concentration of ...

Embodiment 3

[0076] Example 3. Fusion protein purification

[0077] According to the optimized induction conditions, the transformed cells obtained in Example 1 were induced and cultivated in 250 mL LB medium to express the fusion protein of interest. 10000rpm, 4°C, 10min centrifugation to collect the bacterial cells; wash once with PBS, add 10ml of binding buffer NTA0, ice bath, ultrasonic cracking (500W, 5s / 5s), 10000rpm centrifugation for 25min, take the supernatant, filter with a 20μm filter membrane, Automatic loading to AKTA affinity chromatography column,

[0078] bufferA is 20mM Na 3 PO 4 , 500mM NaCl, 40mM imidazole, pH=7.4;

[0079] bufferB is 20mM Na 3 PO 4 , 500 mM NaCl, 500 mM imidazole, pH=7.4.

[0080] Carry out gradient elution successively with the eluent (60mM, 80mM, 100mM, 200mM) containing the following concentrations of imidazole, collect the eluent for electrophoresis detection, use the gel protein analysis software (Quantity one 4.6.2) to calculate the purity, ...

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Abstract

The invention discloses plectasin as well as a gene and a preparation method thereof. A nucleotide sequence of the gene of plectasin is shown in SEQ ID No.1 in a sequence table. The invention discloses a gene sequence of plectasin fusion protein, an amino acid sequence, a recombinant expression vector containing the gene of plectasin and a recombinant expression transformant containing a gene coding the plectasin fusion protein, wherein the gene sequence of the plectasin fusion protein contains enzyme digestion sites of His-tag and TEV enzymes. The preparation method of the plectasin comprises the following steps of: cultivating the recombinant expression transformant; separating and purifying the obtained plectasin fusion protein; adding TEV enzyme and carrying out enzyme digestion; and separating and purifying to obtain plectasin protein. By adopting the preparation method of the plectasin, expression quantity of the plectasin is greatly increased, and the plectasin is simpler and easier to separate and purify. The purified plectasin can effectively inhibit growth of gram positive bacteria, bacillus subtilis and methicillin-resistant staphylococcus epidermidis golden yellow staphylococci.

Description

technical field [0001] The invention belongs to the field of bioengineering, and particularly relates to a plectasin fusion protein and its gene, a recombinant expression vector containing the gene, a recombinant expression transformant, and a preparation method of the plectasin. Background technique [0002] Bacterial drug resistance is widespread in humans, animals, and agricultural crops, and the need for research and development of new antibacterial drugs is imminent. Plectasin is an antibacterial peptide isolated from a fungus, which can fight against bacteria, fungi and viruses. Recently, it has been found that antimicrobial peptides with primary, secondary, and even quaternary structures are similar in spiders, scorpions, flies, mussels, etc. High-purity plectasin can be obtained by adopting the method of bioengineering recombination. In vitro studies have shown that these recombinant plectasins have a good killing effect on various Gram-positive bacteria, including...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K19/00C12N15/62C12N15/70C12N1/21C07K1/36C07K1/22C07K1/20C12R1/19
Inventor 胡又佳谢丽萍梁文张伟朱宝泉
Owner SHANGHAI INST OF PHARMA IND CO LTD
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