CN104491851B - Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein - Google Patents
Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein Download PDFInfo
- Publication number
- CN104491851B CN104491851B CN201410724321.6A CN201410724321A CN104491851B CN 104491851 B CN104491851 B CN 104491851B CN 201410724321 A CN201410724321 A CN 201410724321A CN 104491851 B CN104491851 B CN 104491851B
- Authority
- CN
- China
- Prior art keywords
- portunus trituberculatus
- pacifastin
- ptplc
- trituberculatus miers
- proteinase inhibitor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to an application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein (PtPLC). A recombinant expression product of the portunus trituberculatus PtPLC is used for preparing antibacterial agents, immunopotentiators, feed additives, antiseptics or preservatives. The portunus trituberculatus Pacifastin serine proteinase inhibitor protein has potential application value in the aspects of medicine development, feed additive production and the like; and a foundation is laid for disease control and gene-assisted breeding of the portunus trituberculatus.
Description
Technical field
The invention belongs to technical field of molecular biology, specifically a kind of Portunus trituberculatus Miers Pacifastin types silk ammonia
The application of pepsin inhibitor protein (PtPLC).
Background technology
Serpin is the class protein for having inhibitory activity to serine protease, its by with target enzyme
Stable complex is bound to each other to form, prevents the harmful water of serine protease in body from deactivating, be that biology is exempted from vivo
The important component part of epidemic disease system.According to the similitude of sequence signature, topological structure and function, serpin can
To be divided into 74 families, but the report of its 26S Proteasome Structure and Function is less in the crustaceans such as shrimp crab.
Pacifastin is the new serpin family of a class, is initially 1987 in crayfish
It is found in the hemolymph of Pacifastus leniusculus.Research shows that the gene is existed only in arthropod, at present
Pacifastin related gene has been found in various insects, but only in crayfish and Eriocheir sinensis in crustacean
Middle discovery.The Pacifastin of crayfish is made up of two peptide chains, heavy chain and light chain, and wherein light chain contains one typical half
Cystine pattern of rows and columns Cys-Xaa9-12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2-3-Gly-Xaa3-4-Cys-Thr-Xaa3-
Cys(Pacifastin l ight chain domain,PLD).PLD configuration domain is to the 26S Proteasome Structure and Function of Pacifastin to pass
It is important.
Portunus trituberculatus Miers (Portunus trituberculatus) is the important sea-farming kind of China, but with foster
Grow the continuous expansion of scale and the continuous improvement of cultivation intensive degree, the various diseases caused by bacterium, fungi and virus etc.
Evil is on the rise, and brings about great losses to Portunus trituberculatus Miers aquaculture.Therefore, carry out Portunus trituberculatus Miers immunity epidemic prevention basis to grind
Study carefully, be that more scientific effective approach is found in the preventing and treating of crab disease, prevent and reduce crab disease by improving crab autoimmunity resistance
Generation, be the reliable guarantee for realizing its healthy aquaculture.
Up to the present, it is not related to the report of Portunus trituberculatus Miers Pacifastin type serpins.Cause
This, research immune factor Pacifastin undoubtedly has to understanding the immune defence mechanism of Portunus trituberculatus Miers and carrying out disease control
There is highly important theory and practice meaning, while will be helpful to develop natural drug for the treatment of human diseases.
The content of the invention
It is an object of the invention to provide a kind of Portunus trituberculatus Miers Pacifastin type serpins
Using.
For achieving the above object, the technical solution used in the present invention is：
A kind of application of Portunus trituberculatus Miers Pacifastin types serpin PtPLC, three wart
Swimming crab PtPLC recombination expression products are used for preparing antibacterials, immunopotentiator, feed addictive, preservative or antistaling agent.
The Portunus trituberculatus Miers PtPLC recombination expression products are used for preparing phenoloxidase inhibitors medicine or Gram-negative
The antibacterial medicines of bacterium.
The Gram-negative bacteria is vibrio alginolyticus or pseudomonas aeruginosa.
The Portunus trituberculatus Miers PtPLC recombination expression products are obtained in a conventional manner, the following institute of concrete amino acid sequence
Show,
MRGSHHHHHHGMASELALTSPPFVELPSDPDAPECEGRPLVDRWRKDCNWCSCNEGRVRCSRQLCPEGQ
QDPEPQCEGSPTWKDDCNTCRCAGGRAVCTAKHCDQLGPEQQIVEVQVESAECKEGSRWRVECNWCTCRGGKGACTE
MACLNWDEDQAREDGILECHGSSRWKKDCNWCRCAEGRGFCTKKACPQTGPFDNLPEDATCVPGSRWLVDCNWCGCS
DDGRSSFCTLMACIPGYVHEGPTCEDGSVWKTDDCNICRCIDGMSACTKRLCATPN
Advantage for present invention：
When the present invention is 2.28,4.56,9.11 μM using Portunus trituberculatus Miers PtPLC recombinant expression proteins concentration, in reaction
During 40min, 18.95-72.75% is reached to the inhibitory activity of the broken lysate phenol oxidase of Portunus trituberculatus Miers haemocyte.Restructuring
Albumen PtPLC is to having obvious inhibition in exponential phase and the vibrio alginolyticus of the stage of stable development (4-10h), right with feminine gender
Compare according to group, reached significant difference level (P<0.05).Recombinant protein PtPLC is in exponential phase (3-4,6-7h)
Pseudomonas aeruginosa has obvious inhibition, compared with negative control group, has reached significant difference level (P<0.05).
Gene of the present invention and its recombinant protein can be applied to the mankind related as the production of phenol oxidase class inhibitor medicaments
The treatment of disease, or the production for feed addictive, preservative or antistaling agent etc., it can in addition contain for further studying three warts
Swimming crab immune defence mechanism provides basis, and the disease control for Portunus trituberculatus Miers and gene assist-breeding provide reference.
Description of the drawings
Portunus trituberculatus Miers Pacifastin type serpins of the Fig. 1 for present example variable concentrations
Inhibitory action figure (Tris-HCL of the PtPLC recombinant proteins to Portunus trituberculatus Miers hemocyte lysate supernatant (HLS) phenol oxidase
For negative control).
Fig. 2 is present example Portunus trituberculatus Miers Pacifastin type serpin PtPLC recombinant proteins
The inhibitory action figure of (9.11 μM of final concentration) to vibrio alginolyticus (Tris-HCL is negative control).
Fig. 3 is present example Portunus trituberculatus Miers Pacifastin type serpin PtPLC recombinant proteins
The inhibitory action figure of (9.11 μM of final concentration) to P. aeruginosa (Tris-HCL is negative control).
Specific embodiment
The present invention is further elaborated in the following examples, but the invention is not restricted to this.
Embodiment 1.
The phenol oxidase of Portunus trituberculatus Miers Pacifastin type serpin PtPLC recombinant proteins suppresses examination
Test：
1. the preparation of Portunus trituberculatus Miers hemocyte lysate supernatant (HLS)：Portunus trituberculatus Miers haemocyte is extracted, is rapidly added
To in anti-coagulants, after centrifugation 10min, haemocyte precipitation is obtained.After using 0.1M PBS (pH=7.0) resuspended precipitation, use
The ultrasonic wave of 20kHz/100W is impacted 4 times, and after each 30s, 12,000rpm 4 degree of centrifugation 10min remove precipitation.Supernatant is
For hemocyte lysate supernatant (HLS), 4 degree of environment are put into standby.
2. the measure of Phenoloxidase Activities：By the PtPLC recombinant proteins (final concentration point of 40 μ L HLS and 50 μ L variable concentrations
Wei 2.28,4.56,9.11 μM) it is placed in 96 orifice plates after mixing.By 10 μ L vibrio alginolyticus (OD560=0.4) be added in 96 orifice plates
Incubation at room temperature 5min, the L-dopa solution for being subsequently adding 100 μ L 2mg/ml start reaction.After the reaction 0,5,10,15,20,
30 and 40min use ELIASA (Emax) reading under 490nm wavelength respectively, measure the activity of phenol oxidase.After reaction 20min
PO activity in Portunus trituberculatus Miers HLS reaches plateau, after reaction 40min finds that above-described embodiment recombinant protein PtPLC exists
When final concentration of 2.28,4.56,9.11 μM, the inhibitory activity of the broken lysate phenol oxidase of Portunus trituberculatus Miers haemocyte is reached
18.95-72.75%.
Embodiment 2.
The external vibrio alginolyticus suppression of Portunus trituberculatus Miers Pacifastin type serpin PtPLC recombinant proteins
System test：
1. the culture and preparation of microorganism：Vibrio alginolyticus belongs to vibrionaceae, vibrio, fermented type Gram-negative quarter butt
Bacterium, is connected to " C " or serpentine without gemma, pod membrane, individualism or tail end, is the mesophilic property of thermophilic salt, the raw vibrios in amphimicrobian sea,
It is the important pathogen of Portunus trituberculatus Miers milky disease.
Vibrio alginolyticus TSB culture mediums are to cultivate on 220rpm/min shaking tables in 28 DEG C, rotating speed, make bacteria concentration reach logarithm
During growth period, thalline is diluted with 50mM Tris-HCl (pH=8.0) buffer solution so as to which the clump count in every milliliter of bacterium solution is about 1
×103。
2. recombinant protein PtPLC Antibacterial Activities：The vibrio alginolyticus bacterium solution of 50 μ L exponential phases is taken, equal-volume is added
The PtPLC recombinant proteins (9.11 μM of final concentration) being dissolved in 50mM Tris-HCL (pH=8.0) buffer solution, are incubated under room temperature
30min.Mixed liquor after 20 μ L incubations is taken, is added in 96 orifice plates containing 180 μ L TSB fluid nutrient mediums.In 28 DEG C, rotating speed it is
Cultivate on 220rpm/min shaking tables, every 1h readings under visible ray of the wavelength for 560nm continuously measure 10h.It was found that above-mentioned enforcement
Example recombinant protein PtPLC in exponential phase and the vibrio alginolyticus of the stage of stable development (4-10h) have obvious inhibition (see
Fig. 2), compared with negative control group, significant difference level (P has been reached<0.05).
Embodiment 3.
The external P. aeruginosa of Portunus trituberculatus Miers Pacifastin type serpin PtPLC recombinant proteins
Bacterium suppresses test：
1. the culture and preparation of microorganism：Pseudomonas aeruginosa belongs to pseudomonadaceae, pseudomonad also known as Pseudomonas aeruginosa
Category, non-fermented type gram-Negative bacillus, thalline are elongated and different in size, and there is single flagellum one end of thalline, is to need bacterium, exists
In soil, dust, water, it is a kind of common conditioned pathogen, but non-Portunus trituberculatus Miers pathogen.
Pseudomonas aeruginosa LB culture mediums are to cultivate on 220rpm/min shaking tables in 37 DEG C, rotating speed, reach bacteria concentration
During exponential phase, thalline is diluted with 50mM Tris-HCl (pH=8.0) buffer solution so as to the clump count in every milliliter of bacterium solution
About 1 × 103。
2. recombinant protein PtPLC Antibacterial Activities：Take the bodies such as the P. aeruginosa bacterium solution of 50 μ L exponential phases, addition
Product is dissolved in the PtPLC recombinant proteins (9.11 μM of final concentration) in 50mM Tris-HCL (pH=8.0) buffer solution, incubates under room temperature
Educate 30min.Mixed liquor after 20 μ L incubations is taken, is added in 96 orifice plates containing 180 μ L LB fluid nutrient mediums.In 28 DEG C, rotating speed it is
Cultivate on 220rpm/min shaking tables, every 1h readings under visible ray of the wavelength for 560nm continuously measure 10h.It was found that above-mentioned enforcement
Example recombinant protein PtPLC in exponential phase (3-4,6-7h) pseudomonas aeruginosa have obvious inhibition (see
Fig. 3), compared with negative control group, significant difference level (P has been reached<0.05).
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (1)
1. a kind of application of Portunus trituberculatus Miers Pacifastin types serpin PtPLC, it is characterised in that：
The Portunus trituberculatus Miers PtPLC recombination expression products are used for preparing phenoloxidase inhibitors medicine or Gram-negative bacteria
Antibacterial medicines；
The Gram-negative bacteria is vibrio alginolyticus or pseudomonas aeruginosa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410724321.6A CN104491851B (en) | 2014-12-03 | 2014-12-03 | Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410724321.6A CN104491851B (en) | 2014-12-03 | 2014-12-03 | Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104491851A CN104491851A (en) | 2015-04-08 |
| CN104491851B true CN104491851B (en) | 2017-03-22 |
Family
ID=52933364
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410724321.6A Active CN104491851B (en) | 2014-12-03 | 2014-12-03 | Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104491851B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6771037B2 (en) * | 2015-11-06 | 2020-10-21 | コオペラティ・アヴェベ・ユー・エイ | Fermentation method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104117059A (en) * | 2013-04-28 | 2014-10-29 | 中国科学院海洋研究所 | Application of portunus trituberculatus serine protease gene |
-
2014
- 2014-12-03 CN CN201410724321.6A patent/CN104491851B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104117059A (en) * | 2013-04-28 | 2014-10-29 | 中国科学院海洋研究所 | Application of portunus trituberculatus serine protease gene |
Non-Patent Citations (2)
| Title |
|---|
| The first homolog of pacifastin-related precursor in the swimming crab (Portunus trituberculatus): Characterization and potential role in immune response to bacteria and fungi;Shuangyan Wang;《Fish & Shellfish Immunology》;20111130;第32卷;331-338 * |
| 三疣梭子蟹(Portunus trituberculatus) 丝氨酸蛋白酶抑制剂基因的克隆和功能分析;王双艳;《中国优秀硕士学位论文全文数据库 农业科技辑》;20131215(第S1期);D052-32 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104491851A (en) | 2015-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kavitha et al. | Evaluation of probiotic potential of Bacillus spp. isolated from the digestive tract of freshwater fish Labeo calbasu (Hamilton, 1822) | |
| Abarike et al. | Effects of a commercial probiotic BS containing Bacillus subtilis and Bacillus licheniformis on growth, immune response and disease resistance in Nile tilapia, Oreochromis niloticus | |
| Wang et al. | Amelioration of Cd-induced bioaccumulation, oxidative stress and intestinal microbiota by Bacillus cereus in Carassius auratus gibelio | |
| Zhou et al. | Role and functions of beneficial microorganisms in sustainable aquaculture | |
| Wei et al. | Damage to the membrane permeability and cell death of Vibrio parahaemolyticus caused by phlorotannins with low molecular weight from Sargassum thunbergii | |
| CN103320365A (en) | Fish-sourced aeromonas hydrophila disease antagonistic strain and application thereof | |
| Asaduzzaman et al. | Sodium alginate supplementation modulates gut microbiota, health parameters, growth performance and growth‐related gene expression in Malaysian Mahseer Tor tambroides | |
| CN104491851B (en) | Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein | |
| CN103966122B (en) | One strain Providence and the application in preparation antibacterials thereof | |
| CN103387949B (en) | Scophthalmas maximus fin rot disease antagonistic bacterium and application thereof | |
| Li et al. | Expression and antimicrobial activity of c-type lysozyme in taimen (Hucho taimen, Pallas) | |
| US20060257374A1 (en) | Use of bdellovibrionaceae as an antimicrobial agent | |
| Li et al. | Immunoregulatory effect assessment of a novel melanin and its carboxymethyl derivative | |
| CN106011088A (en) | Establishment method of cLYZ/hTLF double-antibacterial gene recombinant adenovirus, and recombinant adenovirus and application thereof | |
| CN113444696B (en) | Aeromonas hydrophila phage and application thereof | |
| CN102037917B (en) | Application of bdellovibrio sp. leech plasmid bacterium solution in cultivating penaeus monodon | |
| Zhu et al. | Screening of host gut-derived probiotics and effects of feeding probiotics on growth, immunity, and antioxidant enzyme activity of hybrid grouper (Epinephelus fuscoguttatus♀× E. lanceolatus♂) | |
| CN106987543B (en) | A kind of serratia marcescens BSFL-6 in stratiomyiid enteron aisle source and its application | |
| CN105907658A (en) | Peony endogenous Bacillus subtilis and its separation method and use | |
| CN103966121A (en) | Pseudomonas aeruginosa and application of pseudomonas aeruginosa in preparing antibacterial drugs | |
| WO2016037296A1 (en) | Method for producing indigenous probiotocs with immunostimulant activity and use thereof in prophylaxis against flavobacteriosis in salmonids | |
| Devi et al. | Enhancement of growth potentials in freshwater prawn Macrobrachium rosenbergii through supplementation of probiotic diets of Bacillus subtilis and Lactobacillus rhamnosus | |
| Rosland | Evaluation of potential probiotic bacteria for microalgae propagation and Artemia franciscana (Kellog, 1906) bioencapsulation | |
| CN103815138A (en) | Bdellovibrio bacteriovorus nutrient enhancer for holothurian | |
| Zulhisyam et al. | Local (Malaysian) leech as alternative healing treatment and an Islamic perspective |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |