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Crassostrea hongkongensis LysM protein having bacteriostatic activity, and encoding gene and application of crassostrea hongkongensis LysM protein

A Hong Kong oyster and protein technology, applied in Hong Kong oyster LysM protein and its coding gene and application field, can solve problems such as economic loss, threat to the sustainable development of existing industries, and death of oyster culture

Active Publication Date: 2018-11-13
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the 1990s, large-scale deaths of farmed oysters have occurred one after another, which not only caused huge economic losses, but also seriously threatened the sustainable development of the existing industry

Method used

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  • Crassostrea hongkongensis LysM protein having bacteriostatic activity, and encoding gene and application of crassostrea hongkongensis LysM protein
  • Crassostrea hongkongensis LysM protein having bacteriostatic activity, and encoding gene and application of crassostrea hongkongensis LysM protein
  • Crassostrea hongkongensis LysM protein having bacteriostatic activity, and encoding gene and application of crassostrea hongkongensis LysM protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] In Vitro Recombinant Expression and Purification of LysM Protein from Hong Kong Oyster

[0021] The amino acid sequence of the Hong Kong oyster LysM protein is shown in SEQ ID NO.1:

[0022] MSSRKSRQDANNKPYSYQQLGAEVQNSKKSRVYVFGNADVQEDEVVEFEMSDVRSRKGGPKPTQKDEDEQLYYERELTEGDTLRSLSLQYGCPVAEIKRINNMIQDQDFYAYRKIKVPIKKYSFLTETLKTETKSDKPQFNGITVVDETDTENTETDSESVCNMSDPETQRLMIKKSLSIRSQTGLQSKEARRFLRSMDKDLVKICKSAKMRHESLDEVVSLLTNRSIQPIPPPRRKFFGHDCGLTWCSMIGVIVVLAILIPLGILSYLWFTGHFSTHSSAGQNNG

[0023] Length: 286 amino acids

[0024] Type: protein

[0025] Chain type: single chain

[0026] Topology: Linear

[0027] Features: The molecular weight is 32.7kDa, the isoelectric point is 8.16, and its coding sequence has a conserved LysM domain

[0028] Source: Hong Kong Oyster (Crassostrea hongkongensis)

[0029] 1. Construction of prokaryotic expression vector of LysM gene of Hong Kong oyster: (1) design a pair of primers F1 covering ChLysM (LysM gene of Hong Kong oyster) ORF (its nucleot...

Embodiment 2

[0033] Hong Kong oyster LysM protein recognizes peptidoglycan (PGN) from different sources

[0034] (1) Dissolve PGN from three sources of staphylococcus aureus (Sigma, 77140), Bacillus subtilis (Sigma, 69554), and Saccharomyces cerevisiae (Sigma, 72789) in double distilled water (40mg / ml); (2) respectively Pipette 50 μl into a 96-well plate (Costar), and place at room temperature until evaporated to dryness; (3) fix PGN at 60°C for half an hour; (4) add 200μl Tris Buffer containing BSA, 37°C, 2h; (5) discard Supernatant, Tris Bufferwash 4 times, 2min each time; (6) Add purified ChLysM recombinant protein at different concentrations, and incubate at room temperature for 3h; (7) Discard the supernatant, Tris Buffer wash 4 times, 2min each time; (8) Add mouse anti-GST antibody (Abmat, 1:5000), incubate for 2 h; (9) discard the supernatant, Tris Buffer wash 4 times, 2 min each time; (10) add HRP-coupled goat anti-mouse IgG (Abmat, 1 :3000), incubated for 2h; (11) adding HRP subs...

Embodiment 3

[0037] Antibacterial activity of LysM protein from Hong Kong oyster

[0038](1) Vibrio alginolyticus (Vibrio alginolyticus), hemolytic staphylococcus (staphylococcus aureus), staphylococcus aureus (staphylococcus aureus) and Escherichia coli (Escherichia Coli) four kinds of bacteria use LB culture medium at respective suitable temperature, 220rpm, cultivate to During the logarithmic growth phase; (2) Dilute the bacteria with Tris-HCl (50mmol L-1, pH=8.0) so that the number of colonies per milliliter of the bacteria solution is about 1×10 4 CFU; (3) 50 μL of Hong Kong oyster LysM protein (100 ng / μL) was incubated with 50 μL of the above bacterial dilution at 30° C. for 3 h. (4) Take 20 μL of the above mixture in a flat-bottomed 96-well plate (Costar), add 200 μL LB liquid medium to each well, continue culturing, and measure and record the OD600 value of each well every 1 h. (5) Set up BSA protein control group and PBS blank control group. Three repetitions are set for each sa...

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Abstract

The invention discloses a crassostrea hongkongensis LysM protein having a bacteriostatic activity, and an encoding gene and an application of the crassostrea hongkongensis LysM protein. The LysM protein is separated from crassostrea hongkongensis for the first time, PGN is effectively recognized, and the LysM protein has an obvious bacteriostatic effect on the growth of alginolyticus, staphylococcus haemolyticus, staphylococcus aureus and escherichia coli. The LysM protein plays important roles in immunological recognition of the crassostrea hongkongensis and the antibacterial activity, provides evidences for developing broad-spectrum antibiotics and screening an immunopotentiator, and also provides an application guide for the disease resistance and breeding of the shellfish.

Description

Technical field: [0001] The invention belongs to the technical field of applied marine organisms, and in particular relates to a Hong Kong oyster LysM protein with antibacterial activity, its coding gene and its application. Background technique: [0002] Crassostrea hongkongensis, the Hong Kong oyster, is the main economic species cultured in the coastal areas of South my country. It likes to live near river mouths or places with fresh water injection. It is mainly distributed in Guangdong, Guangxi and other places, with an annual output of more than 1.3 million tons and an output value of 60-80 million tons. billion. Compared with other types of oysters, the value of Hong Kong oysters is mainly reflected in their large body, fat meat, and delicious taste. In addition, their market value is much higher than other types of oysters, and they are favored by consumers in Guangdong, Guangxi, Hong Kong, Macao, and some countries in Southeast Asia. However, since the 1990s, large-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70C12N1/21A61K38/17A61P31/04C12R1/19
CPCA61K38/00A61P31/04C07K14/43504C12N15/70
Inventor 李军张扬张跃环肖述向志明马海涛喻子牛
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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