Type two peptidoglycan recognition protein and preparation method and application thereof

A technology for identifying proteins and peptidoglycan, which is applied in the fields of peptide/protein components, biochemical equipment and methods, medical preparations containing active ingredients, etc. Effect

Inactive Publication Date: 2013-06-05
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are very few peptidoglycan recognition proteins with bactericidal effects in fish, and they are only found in zebrafish and flatfish, and have not been reported in other fish species.

Method used

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  • Type two peptidoglycan recognition protein and preparation method and application thereof
  • Type two peptidoglycan recognition protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The type II peptidoglycan recognition protein is shown in the amino acid sequence of SEQ ID No.1 in the sequence table. The sequence listing SEQ ID No.1 is:

[0021] MTSFGVSIFVLGLSCFFTVYSRPAGVHLRNMDSFIRAVQQVEDSNPGISPLALVRALRRTAGHD

[0022] DAMTIHFLGASYNLSDAEVLETAILNASSFSFFDKAIHHIVTDYGEERGVVLTPDGTTVALAPL

[0023] LLG I ELGLKAKMEGTPAVGLFSLTLRTLGLSFLSLQDFPPSYRLGPNGCWDNVDYPKMFKLSQ

[0024] AATLATDAVINGGMDGAILGT

[0025] DLSNLSAHEPPQALSDILKGYYSFILHEGQGLDALTKHVSPRR

[0026] REISRAILEPLDLHSQVMETLALVWKLEKTEWIAFDTGLGKAVKDGLERFVHKYWDCPQIIPRC

[0027] QWGAKAHRGTPPLPLSLPLRFLYVHHTYEPSSPCLSFPTCSRDMRSMQRYHQEDRGWSDIGYSFV

[0028] VGSDGYVYEGRGWNQLGRHTRGHNDVGYGVSIIGNYTATLPSRHAMDLLLRHRLVRCAVDGGGLV

[0029] ANFTIHGHRQVVNYTSCPGDAFFSEIRTWEHFRD

[0030] (a) Sequence features:

[0031] ●Length: 482

[0032] ●Type: amino acid sequence

[0033] ●Chain type: single chain

[0034] ●Topological structure: linear

[0035] (b) Molecule type: protein

[0036] (c) Assumption: No

[0037] ...

Embodiment 2

[0041] The preparation method of type II peptidoglycan recognition protein:

[0042] 1) Construction of plasmid pPGRP2:

[0043] Using American redfish cDNA as a template, PCR amplification was performed with primers PGRPF1 and PGRPR1. The PCR conditions are: 94°C for 60s to pre-denature template DNA, then 94°C for 40s, 55°C for 60s, 72°C for 60s, after 5 cycles, change to 94°C for 40s, 61°C for 60s, 72°C for 60s, and after 25 cycles, repeat at 72°C. ℃ extension reaction 7-10min. After the PCR product was purified, it was ligated with the carrier pBS-T (purchased from "Tiangen Biochemical Technology (Beijing) Co., Ltd.") at room temperature for 2-8 hours, and the ligation mixture was transformed into E. ml Xgal and isopropyl-β-D-thiogalactoside (24ug / ml) were cultured on LB medium for 18-24 hours, and the transformants were screened to extract a plasmid, which was plasmid pBSPGRP1.

[0044] The above-mentioned plasmid pBSPGRP1 and plasmid pET259 (see Zheng, W.J., Hu, Y.H., ...

Embodiment 3

[0051] Bactericidal effects of type II peptidoglycan recognition proteins

[0052] 1) Bacterial plate preparation. Streptococcus iniae, Staphylococcus aureus and Bacillus subtilis (all preserved in CGMCC, numbered CGMCC No.1984, 1.363, 1.460 respectively) were cultured in LB medium to OD 600 0.5, the bacterial solution was mixed with LB containing 0.8% (mass percentage) agar; the mixed solution was poured into a petri dish, and it was a bacterial plate after solidification.

[0053] 2) Detection of the bactericidal effect of the type II peptidoglycan recognition protein. Place the piece of sterile filter paper on the bacterial plate of 1) above. The peptidoglycan recognition protein prepared in Example 2 was diluted to 5uM in PBSZ, and 10ul of the peptidoglycan recognition protein or PBSZ (negative control) was added to the filter paper of the bacterial plate. After the plate was incubated at 28°C for 12-14 hours, it was observed that an obvious inhibition zone was formed a...

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Abstract

The invention relates to the field of molecule microbiology, in particular to a type two peptidoglycan recognition protein (PGRP) of sciaenops ocellatus and a preparation method and application of the type two peptidoglycan recognition protein of the sciaenops ocellatus. PGRP is shown as an amino acid sequence in a sequence table sequence (SEQ) ID number (No.) 1. The preparation method comprises the steps that plasmid p PGRP2 is converted to escherichia coli BL 21 (DE3) to obtain BL21 or the p PGRP 2, the BL21 or the p PGRP 2 after the inducement of isopropyl-beta-D-sulfo-galactoside recombines protein through the purification of affinity chromatography, namely, the protein has bacteriostatic activity. The type two PGRP has the function of specificity fungicide which aims at gram-positive bacterium, so that the growth or the infection of the gram-positive bacterium can be retrained.

Description

technical field [0001] The invention relates to the field of molecular microbiology, in particular to an American redfish type II peptidoglycan recognition protein (peptidoglycan recognition protein, PGRP) and its preparation and application. Background technique [0002] Peptidoglycan (peptidoglycan) is a macromolecule formed by cross-linking heteropolysaccharides of N-acetylglucosamine and N-acetylmuramic acid alternately linked with peptides of different compositions. Peptidoglycan is a major component of many bacterial cell walls, especially in Gram-positive bacteria, where peptidoglycan accounts for 50-80% of the dry weight of the cell wall. Peptidoglycan recognition protein is an innate immune molecule widely present in vertebrates and can specifically recognize peptidoglycan. The peptidoglycan recognition protein recognizes the peptidoglycan of invading pathogenic bacteria, thereby activating Toll and Imd and other immune signaling pathways and mediating phagocytosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/70C12N15/66A61K38/50A61P31/04C12R1/19
Inventor 孙黎李墨非张敏
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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