CN104491851A - Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein - Google Patents
Application of portunus trituberculatus Pacifastin serine proteinase inhibitor protein Download PDFInfo
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- CN104491851A CN104491851A CN201410724321.6A CN201410724321A CN104491851A CN 104491851 A CN104491851 A CN 104491851A CN 201410724321 A CN201410724321 A CN 201410724321A CN 104491851 A CN104491851 A CN 104491851A
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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 application of portunus trytuberculatus Pacifastin type serpin (PtPLC).
Background technology
Serpin is class protein serine protease being had to inhibit activities, it forms stable complex by be combineding with each other with target enzyme, preventing harmful hydrolysis of serine protease in body from activating, is immune important component part in organism.According to the similarity of sequence signature, topological structure and function, serpin can be divided into 74 families, but the report of its 26S Proteasome Structure and Function is less in the crustaceans such as shrimp Eriocheir sinensis.
Pacifastin is the serpin family that a class is new, is at first to be found in the hemolymph of crayfish Pacifastus leniusculus for 1987.Research shows that this gene is only present in arthropod, in various insects, finds Pacifastin related gene at present, but only finds in crayfish and Eriocheir sinensis in crustacean.The Pacifastin of crayfish is made up of two peptide chains, heavy chain and light chain, and wherein light chain contains typical cysteine pattern of rows and columns Cys-Xaa
9-12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa
2-3-Gly-Xaa
3-4-Cys-Thr-Xaa
3-Cys (Pacifastin l ight chain domain, PLD).The 26S Proteasome Structure and Function of PLD domain to Pacifastin is most important.
Portunus trytuberculatus (Portunus trituberculatus) is the important sea-farming kind of China, but along with the continuous expansion of cultivation scale and improving constantly of cultivation intensive degree, the various diseases caused by antibacterial, fungus and virus etc. are on the rise, and bring about great losses to portunus trytuberculatus aquaculture.Therefore, carry out portunus trytuberculatus immunity epidemic prevention basic research, for more scientific effective approach is found in the control of Eriocheir sinensis disease, preventing by improving Eriocheir sinensis autoimmune resistance and reduce the generation of Eriocheir sinensis disease, is the reliable guarantee realizing its healthy aquaculture.
Up to the present, the report about portunus trytuberculatus Pacifastin type serpin is not had.Therefore, research immune factor-Pacifastin to understanding the immune defence mechanism of portunus trytuberculatus and carrying out disease control there is very important theory and practice meaning undoubtedly, the treatment will contributing to developing natural drug and be used for human diseases simultaneously.
Summary of the invention
The object of this invention is to provide a kind of application of portunus trytuberculatus Pacifastin type serpin.
For achieving the above object, the technical solution used in the present invention is:
An application of portunus trytuberculatus Pacifastin type serpin PtPLC, described portunus trytuberculatus PtPLC recombination expression product is for the preparation of antibacterials, immunostimulant, feed additive, antiseptic or antistaling agent.
Described portunus trytuberculatus PtPLC recombination expression product is for the preparation of the antibacterial medicines of phenoloxidase inhibitors medicine or gram negative bacteria.
Described gram negative bacteria is vibrio alginolyticus or Pseudomonas aeruginosa.
Described portunus trytuberculatus PtPLC recombination expression product obtains in a conventional manner, and concrete aminoacid sequence is as follows,
MRGSHHHHHHGMASELALTSPPFVELPSDPDAPECEGRPLVDRWRKDCNWCSCNEGRVRCSRQLCPEGQQDPEPQCEGSPTWKDDCNTCRCAGGRAVCTAKHCDQLGPEQQIVEVQVESAECKEGSRWRVECNWCTCRGGKGACTEMACLNWDEDQAREDGILECHGSSRWKKDCNWCRCAEGRGFCTKKACPQTGPFDNLPEDATCVPGSRWLVDCNWCGCSDDGRSSFCTLMACIPGYVHEGPTCEDGSVWKTDDCNICRCIDGMSACTKRLCATPN
The advantage that the present invention has:
When the present invention utilizes portunus trytuberculatus PtPLC recombinant expression protein concentration to be 2.28,4.56,9.11 μMs, when reacting 40min, 18.95-72.75% is reached to the inhibit activities of the broken lysate phenol oxidase of portunus trytuberculatus hemocyte.Recombiant protein PtPLC has obvious inhibition to the vibrio alginolyticus being in exponential phase and the stage of stable development (4-10h), compared with negative control group, reaches significant difference level (P<0.05).Recombiant protein PtPLC has obvious inhibition to the Pseudomonas aeruginosa being in exponential phase (3-4,6-7h), compared with negative control group, reaches significant difference level (P<0.05).
Gene of the present invention and recombiant protein thereof can be used as the production of phenol oxidase class inhibitor medicaments, be applied to the treatment of mankind's relevant disease, or for the production etc. of feed additive, antiseptic or antistaling agent, basis can also be provided for studying portunus trytuberculatus immune defence mechanism further in addition, and provide reference for the disease control of portunus trytuberculatus and gene assist-breeding.
Accompanying drawing explanation
Fig. 1 be example variable concentrations of the present invention portunus trytuberculatus Pacifastin type serpin PtPLC recombiant protein to portunus trytuberculatus hemocyte lysate supernatant (HLS) phenol oxidase inhibitory action figure (Tris-HCL is negative control).
Fig. 2 is the inhibitory action figure (Tris-HCL be negative control) of example portunus trytuberculatus Pacifastin type serpin PtPLC recombiant protein (final concentration 9.11 μMs) of the present invention to vibrio alginolyticus.
Fig. 3 is the inhibitory action figure (Tris-HCL be negative control) of example portunus trytuberculatus Pacifastin type serpin PtPLC recombiant protein (final concentration 9.11 μMs) of the present invention to P. aeruginosa.
Detailed description of the invention
In the following examples, the present invention is further elaborated, but the present invention is not limited thereto.
Embodiment 1.
The phenol oxidase inhibition test of portunus trytuberculatus Pacifastin type serpin PtPLC recombiant protein:
1. the preparation of portunus trytuberculatus hemocyte lysate supernatant (HLS): extract portunus trytuberculatus hemocyte, join rapidly in anticoagulant, after centrifugal 10min, obtains hemocyte precipitation.After resuspended for precipitation 0.1M PBS (pH=7.0), impact 4 times with the ultrasound wave of 20kHz/100W, after each 30s, 12,000rpm, 4 degree of centrifugal 10min, removing precipitation.Supernatant is hemocyte lysate supernatant (HLS), puts into 4 degree of environment for subsequent use.
2. the mensuration of Phenoloxidase Activities: 40 μ L HLS are mixed with the PtPLC recombiant protein (final concentration is respectively 2.28,4.56,9.11 μMs) of 50 μ L variable concentrations and is placed in 96 orifice plates.By 10 μ L vibrio alginolyticus (OD
560=0.4) be added to incubated at room 5min in 96 orifice plates, the L-dopa solution then adding 100 μ L 2mg/ml starts reaction.After the reaction 0,5,10,15,20,30 and 40min use microplate reader (Emax) reading under 490nm wavelength respectively, record the activity of phenol oxidase.PO activity after reaction 20min in portunus trytuberculatus HLS reaches plateau, after reaction 40min, find that above-described embodiment recombiant protein PtPLC is when final concentration is 2.28,4.56,9.11 μMs, 18.95-72.75% is reached to the inhibit activities of the broken lysate phenol oxidase of portunus trytuberculatus hemocyte.
Embodiment 2.
The external vibrio alginolyticus inhibition test of portunus trytuberculatus Pacifastin type serpin PtPLC recombiant protein:
1. the cultivation of microorganism and preparation: vibrio alginolyticus belongs to vibrionaceae, vibrio, fermented type Gram-negative brevibacterium, without spore, pod membrane, individualism or tail end are connected to " C " or serpentine, for addicted to salt addicted to warm nature, amphimicrobian sea raw vibrio, be the important pathogen of portunus trytuberculatus milky disease.
Vibrio alginolyticus TSB culture medium 28 DEG C, rotating speed is that 220rpm/min shaking table is cultivated, when making bacteria concentration reach exponential phase, with 50mM Tris-HCl (pH=8.0) buffer dilution thalline, the clump count in its every milliliter bacterium liquid is made to be about 1 × 10
3.
2. recombiant protein PtPLC Antibacterial Activity: the vibrio alginolyticus bacterium liquid getting 50 μ L exponential phases, add equal-volume and be dissolved in PtPLC recombiant protein (final concentration 9.11 μMs) in 50mM Tris-HCL (pH=8.0) buffer, incubated at room temperature 30min.Get 20 μ L and hatch rear mixed liquor, be added in 96 orifice plates containing 180 μ L TSB fluid mediums.28 DEG C, rotating speed is that 220rpm/min shaking table is cultivated, every 1h is reading under wavelength is the visible ray of 560nm, continuous measurement 10h.Find that above-described embodiment recombiant protein PtPLC has obvious inhibition (see Fig. 2) to the vibrio alginolyticus being in exponential phase and the stage of stable development (4-10h), compared with negative control group, reach significant difference level (P<0.05).
Embodiment 3.
The P. aeruginosa in vitro inhibition test of portunus trytuberculatus Pacifastin type serpin PtPLC recombiant protein:
1. the cultivation of microorganism and preparation: Pseudomonas aeruginosa is also known as bacillus pyocyaneus, belong to pseudomonadaceae, Rhodopseudomonas, non-fermented type gram negative bacilli, thalline is elongated and different in size, there is single flagellum one end of thalline, is needs bacterium, is present in soil, dust, water, a kind of common conditioned pathogen, but non-portunus trytuberculatus pathogen.
Pseudomonas aeruginosa LB culture medium 37 DEG C, rotating speed is that 220rpm/min shaking table is cultivated, when making bacteria concentration reach exponential phase, with 50mM Tris-HCl (pH=8.0) buffer dilution thalline, the clump count in its every milliliter bacterium liquid is made to be about 1 × 10
3.
2. recombiant protein PtPLC Antibacterial Activity: the Pseudomonas aeruginosa liquid getting 50 μ L exponential phases, add equal-volume and be dissolved in PtPLC recombiant protein (final concentration 9.11 μMs) in 50mM Tris-HCL (pH=8.0) buffer, incubated at room temperature 30min.Get 20 μ L and hatch rear mixed liquor, be added in 96 orifice plates containing 180 μ L LB fluid mediums.28 DEG C, rotating speed is that 220rpm/min shaking table is cultivated, every 1h is reading under wavelength is the visible ray of 560nm, continuous measurement 10h.Find that above-described embodiment recombiant protein PtPLC is to being in exponential phase (3-4, Pseudomonas aeruginosa 6-7h) has obvious inhibition (see Fig. 3), compared with negative control group, reach significant difference level (P<0.05).
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (3)
1. an application of portunus trytuberculatus Pacifastin type serpin PtPLC, is characterized in that: described portunus trytuberculatus PtPLC recombination expression product is for the preparation of antibacterials, immunostimulant, feed additive, antiseptic or antistaling agent.
2. by the application of portunus trytuberculatus Pacifastin type serpin PtPLC according to claim 1, it is characterized in that: described portunus trytuberculatus PtPLC recombination expression product is for the preparation of the antibacterial medicines of phenoloxidase inhibitors medicine or gram negative bacteria.
3., by the application of portunus trytuberculatus Pacifastin type serpin PtPLC according to claim 2, it is characterized in that: described gram negative bacteria is vibrio alginolyticus or Pseudomonas aeruginosa.
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| CN108350403A (en) * | 2015-11-06 | 2018-07-31 | 艾维贝合作公司 | Fermentation |
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| 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 |
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| 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 (5)
| Title |
|---|
| SHUANGYAN WANG: "The first homolog of pacifastin-related precursor in the swimming crab (Portunus trituberculatus): Characterization and potential role in immune response to bacteria and fungi", 《FISH & SHELLFISH IMMUNOLOGY》 * |
| SHUANGYAN WANG: "The first homolog of pacifastin-related precursor in the swimming crab (Portunus trituberculatus): Characterization and potential role in immune response to bacteria and fungi", 《FISH & SHELLfiSH IMMUNOLOGY》, vol. 32, 30 November 2011 (2011-11-30), pages 331 - 338 * |
| 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 * |
| 王双艳: "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", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108350403A (en) * | 2015-11-06 | 2018-07-31 | 艾维贝合作公司 | Fermentation |
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