Application of complement serine protease

A technology of serine protease and complement, applied in the field of molecular biology, can solve the problems such as no report of function, unresearched function and applicability of complement I factor or serine protease, no enlightenment, etc., and achieves the effect of simple preparation process.

Active Publication Date: 2020-02-14
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 t...

Method used

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  • Application of complement serine protease
  • Application of complement serine protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0027] The sequence listing SEQ ID No.1 is:

[0028] RVVGGVPAKPTQIQWQIALEENKKIDCGGAYIGGCWVLTAAHCVRPNPVPPFKVKFSLWRKWSAQDTTDIVPVEDIRIHPKYNAATYENDIALVKLEKLPFKDKCFEDNPAISAVCVPWSTQLFQANHTCSISGWGRTIDGRAAQVLLWANVSLIDNCQRFYKDRFRPGMMCAG

[0029] (a) Sequence features:

[0030] ●Length: 173

[0031] ●Type: amino acid sequence

[0032] ●Chain type: single chain

[0033] ●Topological structure: linear

[0034] (b) Molecule type: protein

[0035] (c) Assumption: No

[0036] (d) Antisense: no

[0037] (e) Original source: flounder cDNA was used as a template, PCR amplification was performed with primers F1 and R1, the...

Embodiment 2

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

[0045] Streptococcus iniae was inoculated in TSB medium and cultured at 28°C until OD 600 About 0.8.

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

[0047] The above bacterial dilutions were mixed with dilutions of serine protease rPoCFI-Tryp or tagged protein rTrx (control group) at different concentrations, and CaCl...

Embodiment 3

[0051] Ability of Serine Protease rPoCFI-Tryp to Inhibit the Growth of Various Bacteria Step 1) Cultivation of Bacteria

[0052] Inoculate Edwardsiella tarda, Vibrio harveii, and Vibrio anguillaris in 5ml liquid LB respectively; inoculate Streptococcus iniae in TSB medium, and culture each strain in the respective medium to OD in a conventional manner 600 about 0.8, then dilute the culture solution to 10 with each new medium 6 CFU / ml. Step 2) Determine the growth curves of different bacteria

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

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Abstract

The invention relates to the field of molecular biology, in particular to an application of serine protease (PoCFI-Tryp) of a flounder complement regulatory protein I factor. The invention relates tothe application of serine protease (PoCFI-Tryp) in the flounder complement regulatory protein I factor in preparation of a bacteriostatic agent. The serine protease disclosed by the invention can be combined with various bacteria, and can obviously inhibit the growth of Edwardsiella tarda, Vibrio anguillarum, Vibrio harveyi and Streptococcus iniae. The obtained protein has the application potential in bacterial infection resistance.

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 flounder complement regulatory protein I factor. Background technique [0002] The complement system is an important immune defense system in the body. It exists widely in serum, interstitial fluid and cell membrane surface. The complement system can be activated by the classical pathway, the alternative pathway and the lectin pathway. The activated products formed have the functions of opsonizing phagocytosis, lysing cells, mediating inflammation, Biological functions such as regulating immune response and clearing immune complexes. For complement activation, there is an extremely complex and tight regulatory mechanism in the body to maintain a stable internal environment. Complement I factor is an important negative regulatory protein of complement. Under the action of cofactor, C3b in serum can be degraded into iC3b, furthe...

Claims

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Application Information

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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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