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Plectasin mutant and its gene, preparation method and use

A technology of plectasin and mutants, applied in the field of bioengineering, can solve the problem of low antibacterial activity

Active Publication Date: 2015-07-29
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Therefore the technical problem to be solved in the present invention is exactly: aiming at the defect that the bacteriostasis activity of existing plectasin fusion protein is low, a kind of new plectasin mutant and its gene, preparation method and application are provided

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  • Plectasin mutant and its gene, preparation method and use
  • Plectasin mutant and its gene, preparation method and use
  • Plectasin mutant and its gene, preparation method and use

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Example 1 Error-prone PCR amplification and construction of mutation library

[0058] The present invention utilizes designed primers and plasmid pYG330 as a template (for the construction method of plasmid pYG330, please refer to the published Chinese patent application: 103374579A), explores error-prone PCR conditions, determines the optimal conditions for error-prone PCR, and constructs a mutant library. Since Mg2+ can stabilize non-complementary base pairs during the PCR process; Mn2+ can enhance the specificity of the polymerase to the template, thus adjusting the concentrations of the two ions can obtain diverse libraries with different mutation frequencies. In addition, lowering the annealing temperature, adopting a PCR amplification program with no hot start, no extension after PCR, and increased cycle times can also increase the mismatch rate. Gained error-prone PCR product is connected with Escherichia coli expression vector pET-32a (+)-TEV, the expression pla...

Embodiment 2

[0066] Screening and Identification of Example 2 Positive Clones

[0067] On the 6 plates obtained in Example 1, 20 monoclonal strains were randomly selected, cultivated at 37° C. for 12 hours, and the results of bacterial liquid PCR verification were as follows: Figure 5 As shown, where lane M is Marker; lanes 1 to 7 are the PCR identification results of different colonies. The obtained clones were sequenced by Shanghai Sangon. The sequencing results showed that when the concentration of Mg2+ in the PCR buffer was 15mM, 18mM and 20mM, base mutations occurred in the obtained PCR, and the results are shown in Table 1.

[0068] Table 1 Different Mg 2+ Sequencing results of error-prone PCR-positive transformants at concentrations

[0069]

[0070] Wherein when the PCR buffer Mg 2+ When the concentration was 15mM, 2 of the transformants obtained had base mutations, one was a transversion (the 92nd base was mutated from C to A), and the other was a nonsense mutation; Mg 2+...

Embodiment 3M-1

[0077] Example 3 Induced expression and preliminary separation and purification of M-1, M-4, M-6, M-7 and plectasin

[0078] The plectasin-expressing strain was used as a control strain (for the preparation method of the plectasin-expressing strain, please refer to the published Chinese patent application: 103374579A), the expression vector of this strain is pYG330, and the expression host is E.coli.BL21( DE3). Inoculate 2.5ml of M-1, M-4, M-6, M-7 and the overnight culture of the above-mentioned plectasomycin expression strains into 250ml of LB medium (containing 100μg / ml Amp), shake culture at 37°C for 2.5h to The OD600 was 0.4-0.6, and 0.8 mM IPTG was added to induce expression for 3 hours, and then the bacteria were collected. The induced culture was centrifuged (8000rpm×10min) to obtain the bacteria. Resuspended with 10ml NTA-0, placed in an ice bath and ultrasonically disrupted the wall for 10min (500W, working for 5s, resting for 5s). The cell wall solution was centr...

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Abstract

The invention discloses a plectasin mutant, a gene for coding the plectasin mutant, and a preparation method and use of the plectasin mutant. The plectasin mutant has an amino acid sequence shown in the formula of SEQ ID No.2 in the sequence table. A nucleotide sequence of the gene for coding the plectasin mutant is shown in the formula of SEQ ID No.3. The invention also discloses a fusion protein of the plectasin mutant and a gene for coding the fusion protein. Compared with the existing plectasin, the prepared plectasin mutant has higher bacteriostatic activity, less plectasin use amount, better bacteriostatic effects and a wide medical application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and particularly relates to a mutant polypeptide of Plectasin and its gene, preparation method and application. Background technique [0002] Finding new antibacterial agents has become an urgent problem. With its unique antibacterial mechanism, broad antibacterial spectrum, resistance to drug resistance, and safe source, defensins have attracted increasing attention and become an ideal substitute for traditional antibiotics. In 2005, Mygind et al. (Mygind PH, Fischer RL, Schnorr KM, et al. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus[J]. Nature, 2005, 437(7061): 975-980.) from saprophytic fungus Plectasin was isolated from Ascomycetes, which was considered to be the first fungal defensin, and then attracted great attention. Schmitz J (Schmitz J, Holzgrabe U. Plectasin-a new peptide antibiotic with high therapeutic potential[J]. Pharm Unserer Zeit, 2010,39(...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C07K19/00C12N15/62C12P21/02A61K38/16A61K47/48A61P31/04
CPCA61K38/00C07K14/37C07K2319/20C12N15/70
Inventor 胡又佳谢丽萍张琪陈习平朱宝泉龚桂花
Owner SHANGHAI INST OF PHARMA IND
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