Fused antibacterial peptide and preparation method thereof

An antibacterial peptide, pet-22b-yselp technology, which is applied in the field of fusion antibacterial peptides and their preparation, can solve the problems such as the lack of lethality of host bacteria, and achieve the effect of solving large-scale application.

Inactive Publication Date: 2015-03-25
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a fusion antibacterial peptide and its preparation method. The obtained fusion antibacterial peptide YSELP can

Method used

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  • Fused antibacterial peptide and preparation method thereof
  • Fused antibacterial peptide and preparation method thereof
  • Fused antibacterial peptide and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the preparation of fusion antimicrobial peptide

[0045] 1) Construction of pET-22b-YSELP expression vector: According to the amino acid sequence of the antibacterial peptide YFGAP (as shown in SEQ ID NO.4), the gene sequence of the antibacterial peptide YFGAP was designed with E.coli standard codon optimization, and the gene sequence The 5' end introduces the Nde I restriction site, and the 3' end introduces the EcoR I restriction site, and chemically synthesizes the gene sequence shown in SEQ ID NO.5; the synthesized gene is double-digested with Nde I and EcoR I respectively The sequence was connected to the pUC19 plasmid that had been digested with the same double restriction enzymes by T4 DNA ligase and transformed into E.coli DH5α competent cells (refer to the "Molecular Cloning Experiment Guide" for specific transformation conditions), and the screened positive clones were provided by Sangon Bioengineering (Shanghai) Co., Ltd. sequenced, and the corr...

Embodiment 2

[0054] Embodiment 2: Identification of expression vector pET-22b-YSELP

[0055] The operation is the same as step 1) of Example 1, transform pET-22b-YSELP into E.coli BL21(DE3) competent cells, apply LB plates containing 50 μg / mL ampicillin (Amp) to screen positive clones, and pick Insert a single colony into LB liquid medium, culture at 37°C, 200r / min for 12 hours, extract plasmid DNA (i.e. expression vector pET-22b-YSELP), and verify by Nde I and Bgl I double enzyme digestion, the results are as follows Image 6 As shown, the results of agarose gel electrophoresis show that the molecular weight of the enzyme-cut bands is consistent with the size of the carrier gene, and the three bands are 2583, 2050, and 1502bp respectively; the sequencing results of Sangon Bioengineering (Shanghai) Co., Ltd. also show that the gene sequence is correct , the recombinant expression vector was constructed successfully.

Embodiment 3

[0056] Example 3: Effect of YSELP expression on the growth of host bacteria E.coli BL21(DE3)

[0057] E.coli BL21(DE3) / pET-22b-YSELP was inoculated into LB medium containing 100μg / mL ampicillin at a ratio of 1:100, cultured overnight at 37°C, 200r / min, and inoculated into TB medium, 37°C, 200r / min Cultivate for 4 hours, add IPTG to induce, take the bacterial solution every 1 hour (every 2 hours after 12 hours) to measure the OD value at 600nm; use uninduced E.coli BL21(DE3) / pET-22b-YSELP as the control.

[0058] The result is as Figure 7 As shown, the growth of the host strain E.coli BL21(DE3) was inhibited after adding IPTG to induce expression. After 8 hours of culture, the turbidity of the bacterial solution began to decrease. After 12 hours, the turbidity of the bacterial solution gradually increased, and the concentration of the bacteria increased. E.coli BL21(DE3) grew rapidly again, indicating that the expression of YSELP protein did not completely kill the host itsel...

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Abstract

The invention discloses a fused antibacterial peptide and a preparation method thereof. According to the amino acid sequence of the antibacterial peptide YFGAP, the gene sequence of the amino acid sequence is optimally designed and connected to pUC19 to obtain pUC19-YFGAP; the pUC19-YFGAP is connected to pET-22b-S-ELPs to obtain pET-22b-YSELP; the pET-22b-YSELP is transformed to E.coli BL21 (DE3) competent cells to obtain E.coli BL21 (DE3)/pET-22b-YSELP; inoculating and culturing are performed and IPTG is added to induce the expression of the YSELP. The fused antibacterial peptide YSELP has an inhibition effect on a plurality of testing strains without being cut off from the tag protein, and does not kill host bacteria; as a result, the problem of large-scale application of the existing antibacterial peptides is expected to be solved.

Description

technical field [0001] The invention relates to a fusion antibacterial peptide and a preparation method thereof. Background technique [0002] Aquaculture is an important pillar of our country's agricultural and rural development. Since the 1970s, the world's aquaculture production has grown rapidly, and its proportion in the aquaculture industry is also increasing. There are also huge development potentials for aquaculture in my country. There are two main types of freshwater aquaculture: one is pond intensive culture, which uses bait and fertilization to obtain high yields, and mixes various fish with different feeding habits to give full play to the productivity of the water body. The other type is stocking in large and medium-sized waters such as lakes, reservoirs, river ditches, and rice fields. In addition to the traditional carps, the freshwater aquaculture objects also include non-crucian carp, white crucian carp, rainbow trout, coho salmon, giant giant rosenbergii,...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/14C12N15/62C12N15/70
Inventor 李夏兰张光亚李慧陈培钦葛慧华
Owner HUAQIAO UNIVERSITY
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