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Apostichopus-japonicus glucan binding protein and preparation method and application thereof

A protein and dextran-binding technology, applied in the fields of molecular biology and genetic engineering, can solve problems that plague the industry, less research on macromolecular antibacterial proteins, and adverse effects on the quality and safety of sea cucumber products and the healthy development of the food industry, so as to promote Healthy effect

Active Publication Date: 2019-07-30
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the disease problems encountered in the breeding process have always been an important restrictive factor that plagues the further development of the industry.
However, the inventors found that there are currently more studies on small-molecule antibacterial peptides that are easier to synthesize, but less research on macromolecular antibacterial proteins. The quality and safety of sea cucumber products and the healthy development of related food industries

Method used

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  • Apostichopus-japonicus glucan binding protein and preparation method and application thereof
  • Apostichopus-japonicus glucan binding protein and preparation method and application thereof
  • Apostichopus-japonicus glucan binding protein and preparation method and application thereof

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preparation example Construction

[0039] In yet another specific embodiment of the present invention, a method for preparing glucan-binding protein AJ-GBP is provided, comprising the following steps:

[0040] (1) Using the imitation japonicus japonicus cDNA as a template, carry out PCR amplification with primers F1 and R1, and set aside;

[0041] (2) After the PCR product was purified, it was ligated with the Escherichia coli expression vector pEASY-E2 vector, the ligated product was transformed into Escherichia coli, and the recombinant was identified by sequencing;

[0042] (3) Transfer the above expression vector pEASY-AJ-GBP into Trans BL21(DE3)pLysS chemically competent cells, screen the transformants, inoculate the transformants in LB medium containing ampicillin, induce expression with IPTG, and then use Ultrafiltration tubes were used to purify the recombinant protein to obtain the glucan-binding protein AJ-GBP.

[0043] The primers are respectively:

[0044] F1: 5'-ATGACCGCCGGTGGTGGAGG-3' (SEQ ID NO...

Embodiment 1

[0049] Example 1: Prokaryotic recombinant expression of A. japonicus japonicus AJ-GBP coding region

[0050] Specific steps are as follows:

[0051] 1. Construction of prokaryotic recombinant expression vector

[0052] The present invention adopts pEASY-E2 prokaryotic expression vector produced by Beijing TransGen Biotech Co., Ltd. The entire coding region of AJ-GBP was amplified by PCR with specific primers. The PCR reaction program was set as follows: ① 94 ① pre-denaturation 4 min; ② 94 ① denaturation 30 s; ③ 52 ① renaturation 30 s; ④ 72 ① extension 1 min; Perform steps ②③④ for 40 cycles. The PCR product was purified and recovered, and connected to the pEASY-E2 vector. The ligation product was transformed into Escherichia coli Trans BL21(DE3)pLysS, and 5 to 10 individual colonies were selected and inoculated in LB liquid medium, and sent to a sequencing company for sequencing to verify the correctness of the reading frame. The specific primers were:

[0053] F1:5'-ATGAC...

Embodiment 2

[0065] Example 2: Bacterial agglutination activity analysis of the prokaryotic recombinant protein imitating the AJ-GBP coding region of Apostichopus japonicus

[0066] To Bacillus subtilis, Staphylococcus aureus, Curtobacterium luteum (G + bacteria) and Vibrio anguillarum, Escherichia coli, Pseudomonas aeruginosa (G - bacteria) agglutination test

[0067] Inoculate Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Brevibacterium luteus into LB liquid medium, and cultivate to OD at 37① 600 When the value reached 0.6, Vibrio anguillarum was cultivated to OD at 28① with TCBS medium 600 value to 0.6, then diluted to OD with sterile saline 600 The value is 0.001, then centrifuged at 5000rpm for 10 minutes to collect the bacterial pellet, washed three times with TBS buffer and resuspended to adjust the bacterial concentration to 2×10 9 cells / mL. Add 75 μL of recombinant protein (about 600 μg / ml) to a 96-well plate, then add 30 μL of bacter...

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Abstract

The invention belongs to the technical field of molecular biology and gene engineering, and specifically discloses an apostichopus-japonicus glucan binding protein and a preparation method and application thereof. Through the in-vitro recombinant expression technology, the apostichopus-japonicus glucan binding protein (AJ-GBP) is obtained for the first time, and preferably the amino acid sequenceof the apostichopus-japonicus glucan binding protein (AJ-GBP) is shown in a sequence table SEQIDNO.1. The recombinant protein has an agglutination effect on bacillus subtilis, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, curtobacterium luteum and vibrio anguillarum, can inhibit bacillus subtilis, escherichia coli, curtobacterium luteum and vibrio anguillarum, and has potentialapplication value in the fields of development of novel broad-spectrum antibiotics and feed additives.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to a sea cucumber-imitated glucan-binding protein and its preparation method and application. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Apostichopus japonicus belongs to the phylum Echinodermata, the class Holothuroidea, the order Aspidochirotida, and the family Stichopodidae. It is known as "Ginseng in the Sea" and has high nutritional value. and medical value. It is widely distributed in the waters near Shandong, Liaoning and Hebei in my country. In recent years, with the improvement of people's social living conditions and...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/70C12N1/21A23K20/147A61K38/17A61P31/04
CPCA23K20/147A61K38/00A61P31/04C07K14/43504C12N15/70
Inventor 孔令明雷一萱丛海燕
Owner SHANDONG UNIV
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