Application of a kind of complement serine protease

A serine protease and complement technology, applied in the field of molecular biology, can solve the problems of unreported functions, no enlightenment, unresearched functions and applicability of complement I factor or serine protease, etc., and achieves the effects of no potential safety hazards and simple preparation process.

Active Publication Date: 2022-07-26
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Complement I factors have been found in various fish species, but there are no reports on the functions of the complement-fixing domain, the regulatory domain, and especially the serine protease, so there is no suggestion for this application
And the function and applicability of complement I factors or serine proteases in flounder have not yet been studied

Method used

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  • Application of a kind of complement serine protease
  • Application of a kind of complement serine protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Preparation of Serine Protease Recombinant Protein of Complement Regulating Protein I Factor (1) Construction of Serine Protease Expression Plasmid pEtPoCFI-Tryp The flounder complement serine protease of the present invention is obtained by prokaryotic protein expression system, and its sequence is in SEQ ID No. 1 of the sequence table amino acid sequence.

[0027] Sequence Listing SEQ ID No.1 is:

[0028] RVVGGVPAKPTQIQWQIALEENKKIDCGGAYIGGCWVLTAAHCVRPNPVPFKVKFSLWRKWSAQDTTDIVPVEDIRIHPKYNAATYENDIALVKLEKLPFKDKCFEDNPAISAVCVPWSTQLFQANHTCSISGWGRTIDGRAAQVLLWANVSLIDNCQRFYKDRFRPGMMCAG

[0029] (a) Sequence features:

[0030] ●Length: 173

[0031] ●Type: amino acid sequence

[0032] ●Chain type: single chain

[0033] ●Topology: Linear

[0034] (b) Molecular type: protein

[0035] (c) Assumption: No

[0036] (d) Antonym: No

[0037] (e) The original source: the flounder cDNA was used as the template, and the primers F1 and R1 were used for PCR amplification. The PCR prod...

Embodiment 2

[0044] Detection of the ability of recombinant protein containing serine protease (rPoCFI-Tryp) to bind to various bacteria: different bacteria were inoculated in 5ml liquid LB medium and cultured to OD 600About 0.8, among them, Edwardella lentus, Vibrio harveii, Vibrio eel, and Pseudomonas fluorescens were cultured at 28°C; Escherichia coli, Micrococcus luteus, Staphylococcus aureus, and Bacillus subtilis were cultured at 37°C nourish;

[0045] Inoculate Streptococcus iniae in TSB medium at 28°C to OD 600 about 0.8.

[0046] Dilute each of the above bacteria with coating solution to 10 8 CFU / ml, as bacterial dilution, the purified protein in Example 1 above was diluted in PBS to 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, namely Diluent of rPoCFI-Tryp.

[0047] The above bacterial dilutions were mixed with various concentrations of dilutions of the serine protease rPoCFI-Tryp or the tagged protein rTrx (control) and added with CaCl at a final concentration...

Embodiment 3

[0051] The ability of serine protease rPoCFI-Tryp to inhibit the growth of various bacteria Step 1) Culture of bacteria

[0052] Edwardsiella lentus, Vibrio harveii, and Vibrio eel were inoculated in 5ml of liquid LB; Streptococcus iniae was inoculated in TSB medium, and each strain was cultured to OD in their respective medium in a conventional manner 600 about 0.8, and then the culture medium was diluted to 10 with each new medium. 6 CFU / ml. Step 2) Determine the growth curve of different bacteria

[0053] The purified protein in the above Example 1 was diluted to 70 μg / ml in PBS, which was the rPoCFI-Tryp dilution solution. The above bacterial dilution solutions were mixed with the serine protease rPoCFI-Tryp dilution solution or the tagged protein rTrx (control group), and added. CaCl at a final concentration of 50 μM 2 , as for culturing in a 28°C incubator, measure the absorbance at 600 nm every hour, and draw the growth curves of different bacteria (see figure 2 )....

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Abstract

The invention relates to the field of molecular biology, in particular to the application of a serine protease (PoCFI-Tryp) of a flounder complement regulatory protein I factor. The application of serine protease (PoCFI‑Tryp) in the complement regulatory protein I factor of flounder in the preparation of bacteriostatic agents. The serine protease of the present invention can be combined with various bacteria, and can significantly inhibit the growth of Edwardsiella lentus, Vibrio eel, Vibrio harvey and Streptococcus iniae. The obtained protein has application potential in anti-bacterial infection.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of a serine protease (PoCFI-Tryp) of a flounder complement regulatory protein I factor. Background technique [0002] The complement system is an important immune defense system in the body. It is widely present on the surface of serum, tissue fluid and cell membrane. The complement system can be activated by the classical pathway, the alternative pathway and the lectin pathway. Biological functions such as regulation of immune responses and clearance of immune complexes. For complement activation, there are extremely complex and tight regulatory mechanisms in the body to maintain the stability of the internal environment. Complement I factor is an important complement negative regulator. Under the action of cofactors, C3b in serum can be degraded to iC3b, further degraded into C3c and C3d, preventing the formation of C3 and C5 convertases, and avoiding host immu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/48A61P31/04C12N9/64C12N15/70
CPCA61K38/482C12Y304/21106C12N9/6405C12N15/70A61P31/04
Inventor 李墨非贾贝贝孙黎
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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