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Apostichopus japonicus peptidoglycan recognition protein with sterilization activity as well as preparation method and application of apostichopus japonicus peptidoglycan recognition protein

A technology for identifying proteins and bactericidal activity, which is applied in the field of molecular biology and can solve problems that plague the industry

Active Publication Date: 2017-05-31
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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.
At present, there is no report on the peptidoglycan recognition protein of sea cucumber

Method used

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  • Apostichopus japonicus peptidoglycan recognition protein with sterilization activity as well as preparation method and application of apostichopus japonicus peptidoglycan recognition protein
  • Apostichopus japonicus peptidoglycan recognition protein with sterilization activity as well as preparation method and application of apostichopus japonicus peptidoglycan recognition protein
  • Apostichopus japonicus peptidoglycan recognition protein with sterilization activity as well as preparation method and application of apostichopus japonicus peptidoglycan recognition protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: The prokaryotic recombinant expression of the AJ-PGRP coding region of A. japonicus japonicus, the specific steps are as follows:

[0023] 1. Construction of prokaryotic recombinant expression vector

[0024] The present invention adopts pEASY-E1 prokaryotic expression vector produced by Beijing TransGen Biotech Co., Ltd. The entire coding region of AJ-PGRP was amplified by PCR with specific primers. The PCR reaction program was set as follows: ①pre-denaturation at 94°C for 4 min, ②denaturation at 94°C for 30 s; ③refolding at 52°C for 30 s; ④extension at 72°C for 1 min. ⑤ 72 ℃ final extension 10min. Perform steps ②③④ for 40 cycles. The PCR product was purified and recovered, and connected to the pEASY-E1 vector. The ligation product was transformed into Escherichia coli Trans BL21(DE3), and 5 to 10 single colonies were selected and inoculated in LB liquid medium, and sent to the sequencing company for sequencing to verify the correctness of the reading fr...

Embodiment 2

[0035] Example 2: Bacterial agglutination activity analysis of the prokaryotic recombinant protein imitating the AJ-PGRP coding region of Apostichopus japonicus

[0036] To Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcusaureus) (G + bacteria) and Vibrio splendidus (Vibrio splendidus), Escherichia coli (G - bacteria) agglutination test

[0037] Inoculate Bacillus subtilis, Staphylococcus aureus, and Escherichia coli into LB liquid medium, and cultivate them at 37°C until the OD value reaches 0.6, and culture Vibrio splendidus at 25°C until the OD value reaches 0.6, and then use normal saline Dilute to an OD value of 0.001, then centrifuge at 5000rpm for 10 minutes to collect the bacterial pellet, and use 10mM ZnCL 2 Wash twice with TBS buffer and resuspend to adjust the bacterial concentration to 2×10 9 cells / mL. Add 20 μL of recombinant protein to 96-well plates, then add 10 μL of bacterial solution, and incubate at room temperature for 1 h, an...

Embodiment 3

[0039] Example 3: Antibacterial activity analysis of AJ-PGRP prokaryotic recombinant protein of imitation japonicus japonicus

[0040] Staphylococcus aureus, Bacillus subtilis (G + bacteria) and Vibrio splendidus, Escherichia coli (G - Bacteria) Growth Inhibition Test

[0041]Inoculate Bacillus subtilis, Staphylococcus aureus and Escherichia coli into LB liquid medium, cultivate at 37°C until the OD value reaches 0.6, inoculate Vibrio splendidus into TCBS medium and cultivate at 25°C until the OD value reaches 0.6, take 200uL for culture The solution was added to 35ML LB solid medium that had been sterilized at high temperature and cooled to 50°C, and poured into a 90mm glass plate. After it was completely solidified, two holes with a diameter of 5mm were punched with a puncher, and 45uL The empty vector expression protein and recombinant protein (containing 20uM ZnCl2). The plates were incubated at 30°C for 48 hours. It was observed that an obvious bacteriostatic zone was...

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Abstract

The invention belongs to the field of molecular biology and the technical field of genetic engineering, and in particular discloses apostichopus japonicus peptidoglycan recognition protein with sterilization activity as well as a preparation method and application of the apostichopus japonicus peptidoglycan recognition protein. The apostichopus japonicus peptidoglycan recognition protein AJ-PGRP is obtained for the first time by utilizing an in-vitro recombination expression technology; the apostichopus japonicus peptidoglycan recognition protein (AJ-PGRP) is shown as an amino acid sequence in a sequence table of SEQ ID NO. 1. The recombinant protein has agglutination effect on staphylococcus aureus and escherichia coli, and has inhibition effect on the staphylococcus aureus, the escherichia coli, vibro splendidus and bacillus subtilis, and has potential application value in the aspects of developing novel broad-spectrum antibacterial drugs, feed additives and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology and genetic engineering technology, and specifically relates to a peptidoglycan recognition protein imitating sea cucumber with bactericidal activity, its preparation method and application. Background technique [0002] Apostichopus japonicus belongs to the phylum Echinodermata, the class Holothuroidea, the order Aspidochirotida, and the family Stichopodidae. It is mainly distributed in the Northwest Pacific coast at 35°-44° north latitude, Shandong Province. , Liaoning and Hebei are the main breeding areas of imitation sea cucumber in my country. The imitation sea cucumber ranks first among the "Eight Treasures of the Sea" and has high nutritional and medical value. In recent years, with the improvement of people's social living conditions and health awareness, the market demand for imitation sea cucumbers has increased year by year, which has continuously stimulated the scale of farming. Theref...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/70A61K38/17A61P31/04A23K20/147
CPCA61K38/00C07K14/43504C12N15/70C12N2800/101
Inventor 孔令明丛海燕
Owner SHANDONG UNIV
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