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Recombinant plectasin as well as expression and purification method and application thereof

A technology of lectin and recombinant bacteria, applied in the biological field, can solve the problems of increasing the difficulty of curing diseases, patients and social and economic burden, and achieve the effects of large antibacterial spectrum, good bacteriostatic effect and wide antibacterial range

Active Publication Date: 2015-01-07
陈新
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] With the widespread use of antibiotics in clinical practice, the problem of bacterial resistance is becoming more and more serious, increasing the difficulty of curing diseases and bringing serious economic burdens to patients and society

Method used

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  • Recombinant plectasin as well as expression and purification method and application thereof
  • Recombinant plectasin as well as expression and purification method and application thereof
  • Recombinant plectasin as well as expression and purification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 recombine Plectasin prokaryotic recombine Construction of expression bacteria

[0038] (1) Design the nucleotide sequence of Plectasin as shown in SEQ ID NO.1.

[0039] (2) Construction of prokaryotic expression vector of recombinant plectasin

[0040] 1) Design and synthesis of plectasin nucleotide sequence as shown in SEQ ID NO.3, which was synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0041] 2) Digestion ligation

[0042] The synthetic fragment (shown as SEQ ID NO.3) and the expression vector pE-SUMO were double digested with Bsa I and Xba I and then cloned to the same double digestion. The enzyme digestion system is as follows:

[0043]

[0044] Digested at 37°C for 3 hours, electrophoresed on a 1% agarose gel and recovered using a gel recovery kit. After recovery, the target gene and the expression vector were ligated overnight at 16°C with T4 DNA ligase at a ratio of 3:1 to construct the recombinant expression vector pE-SUMO-Plecta...

Embodiment 2

[0052] Example 2 Plectasin fusion protein (recombinant plectasin) induced expression of

[0053] Pick the positive recombinant expression strain BL21(DE3) verified above and inoculate it in 500ml LB medium, cultivate it on a shaker at 37°C until OD600=0.5, and optimize it by orthogonal experiments with different concentrations of IPTG, different induction temperatures, and different induction times. The induction condition was 30°C, the rotational speed was 180rpm, the inducer used for induction expression was IPTG with a final concentration of 0.05mM, and the induction time was 6h to obtain a higher fusion protein, ie recombinant Plectasin.

Embodiment 3

[0054] Example 3 Plectasin fusion protein (recombinant plectasin) purification of

[0055] Collect the cells by centrifugation, resuspend the cells in 50ml crushing buffer SUMO1, and crush the cells by ultrasonic in an ice bath. Centrifuge at 14,000 rpm for 15 min at 4°C, collect the supernatant, and filter through a 0.22 μm filter. Use 15% SDS-PAGE to detect the protein distribution in the ultrasonic supernatant and precipitate, the protein detection results are as follows figure 2 As shown, M in the figure is the protein molecular weight standard; 1 is the total protein of the non-induced bacteria; 2 is the total protein of the induced bacteria; 3 is the supernatant of the induced bacteria sonicated; 4 is the sonicated precipitate of the induced bacteria. It can be seen that the fusion protein mainly exists in the supernatant with a molecular weight of about 16.7kDa. The total protein content in the ultrasonic supernatant was determined by Bradford method, and the gel...

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Abstract

The invention discloses a recombinant plectasin as well as an expression and purification method and application thereof. The amino acid sequence of the recombinant plectasin is as shown in SEQIDNO.2. The recombinant plectasin is prepared by sequentially carrying out enrichment culture, inductive expression culture and centrifugalization on a recombinant strain containing a gene for encoding an amino acid; sequentially taking, cracking and centrifugalizing thalli, so that a cracked supernatant is obtained; and purifying the cracked supernatant, so that the recombinant plectasin is obtained. The obtained recombinant plectasin is subjected to enzyme digestion by enzyme SUMOpro, and then the obtained product is dialyzed, so that plectasin can be obtained. According to the invention, through the optimization on the expression and purification method of recombinant plectasin, a large amount of high-purity and high-activity plectasin can be obtained, and the obtained plectasin is good in antibacterial effect, wide in antibacterial range, and large in antibacterial spectrum.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant plectasin and its expression and purification method and application. Background technique [0002] With the widespread use of antibiotics in clinical practice, the problem of bacterial resistance is becoming more and more serious, which increases the difficulty of curing diseases and brings serious economic burden to patients and society. Especially in recent years, the number of "super bacteria" has gradually increased, making it extremely urgent to develop a new generation of safe and efficient antibacterial agents while rationally using drugs. [0003] Antimicrobial peptides are small molecular polypeptides with biological activity induced by organisms, and most of them have the characteristics of strong alkalinity, thermal stability and broad-spectrum antibacterial properties. So far, researchers have discovered and isolated peptides with antibacterial activity from...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C07K1/22C07K1/16C12N15/31C12N15/70C12N1/21C12P21/02C12R1/19
CPCC07K14/37
Inventor 陈新李凌史家玮温耀安石羽余楠
Owner 陈新
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