CN108003233A - Long oyster domain protein containing DM9 CgDM9CP-2, preparation method and application - Google Patents

Long oyster domain protein containing DM9 CgDM9CP-2, preparation method and application Download PDF

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Publication number
CN108003233A
CN108003233A CN201711418771.2A CN201711418771A CN108003233A CN 108003233 A CN108003233 A CN 108003233A CN 201711418771 A CN201711418771 A CN 201711418771A CN 108003233 A CN108003233 A CN 108003233A
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cgdm9cp
seq
long oyster
protein containing
domain protein
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CN108003233B (en
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宋林生
刘宇
王伟林
宋小瑞
王玲玲
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Dalian Ocean University
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Dalian Ocean University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
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  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention discloses a kind of long oyster domain protein containing DM9 CgDM9CP 2, and amino acid sequence is as shown in SEQ ID NO.1.Preparation method carries out in accordance with the following steps successively：With specific primer P1 and P2 to long oysterCgDM9CP‑2Gene coding region fragment carries out PCR amplification, and the DNA sequence dna of the primer P1 is as shown in SEQ ID NO.2, and the DNA sequence dna of the primer P2 is as shown in SEQ ID NO.3；Pcr amplification product and pET 30a carriers are passed throughNde1WithXho1Connected, converted, sequencing identification recon by ligase after digestion；Above-mentioned recon is transferred to Escherichia coliTransetta（DE3）Fiber differentiation is carried out in expression bacterial strain, then purifying, renaturation, that is, obtain the recombinant protein with amino acid sequence in sequence table SEQ ID NO.1.Long oyster domain protein containing DM9 CgDM9CP 2, which can be applied to prepare, suppresses Pichia yeast medicine.

Description

Long oyster domain protein containing DM9 CgDM9CP-2, preparation method and application
Technical field
The invention belongs to technical field of molecular biology, more particularly to a kind of long oyster domain protein containing DM9 CgDM9CP-2, preparation method and application.
Background technology
Long oyster is a kind of important sea-farming shellfish.Since long oyster lacks adaptive immunity system of defense, mainly Rely on inherent immunity system and resist infecting for exogenous pathogen microorganism, therefore a variety of diseases exist caused by bacterium, fungi and virus Constantly broken out in the cultured population of long oyster, cause huge economic loss.Albumen comprising DM9 domains is first in drosophila In be found.Afterwards, Magalhaes etc. is found that some toxin with DM9 domains in fish, and is named as " natterin ", this toxoid contain the DM9 domains and C- ends toxin/bacteriotoxin at N- ends（ETX/MTX2）Domain. Natterin can produce cytotoxic effect in tissue, so as to cause meronecrosis, oedema, local pain etc. existing As.Newest research shows that natterin has the function of perforin.In recent decades, more and more contain DM9 domains Protein（DM9CPs）It is found in including invertebrates such as arthropod, flatworms, and proves that DM9CPs joins With the immune response of body.For example, in anopheles costalis, a kind of DM9CPs of entitled " causing plasmodium salivary gland to react " (PRS1), the expression quantity on salivary gland lateral lobe plasmodium invade after significantly raise, and with infection sporozoite quantity increase and Increase.
But not on long oyster domain protein containing DM9 CgDM9CP-2, preparation method and making so far The standby relevant report for suppressing to apply in Pichia yeast medicine.
The content of the invention
The present invention is to solve the above-mentioned technical problem present in the prior art, there is provided a kind of long oyster structure containing DM9 Domain PROTEIN C gDM9CP-2, preparation method and application.
The present invention technical solution be：A kind of long oyster domain protein containing DM9 CgDM9CP-2, it is characterised in that： Amino acid sequence is as shown in SEQ ID NO.1.
A kind of preparation method of above-mentioned long oyster domain protein containing DM9 CgDM9CP-2, it is characterised in that successively according to such as Lower step carries out：
A. with specific primer P1 and P2 to long oysterCgDM9CP-2Gene coding region fragment carries out PCR amplification, the primer P1 DNA sequence dna as shown in SEQ ID NO.2, the DNA sequence dna of the primer P2 is as shown in SEQ ID NO.3；
B. pcr amplification product and pET-30a carriers are passed throughNde1WithXho1Connected, converted by ligase after digestion, sequencing mirror Determine recon；
C. above-mentioned recon is transferred to Escherichia coliTransetta（DE3）Fiber differentiation is carried out in expression bacterial strain, then purifying, Renaturation, that is, obtain the recombinant protein with amino acid sequence in sequence table SEQ ID NO.1.
A kind of above-mentioned long oyster domain protein containing DM9 CgDM9CP-2 answering in suppression Pichia yeast medicine is prepared With.
The present invention obtains the PROTEIN C gDM9CP-2 of long oyster domain containing DM9 using in-vitro recombination expression technology, this is heavy Histone have with reference to Mannose, LPS and PGN biological activity, while outside body to Pichia yeast have it is stronger Inhibitory action.The PROTEIN C gDM9CP-2 of long oyster domain containing DM9 of the present invention as a kind of effective pattern recognition receptors, There is application value preparing antimicrobial DP finish, novel immune preparation and feed addictive etc..
Brief description of the drawings
Fig. 1 is the combination activity of long oyster domain protein containing the DM9 CgDM9CP-2 of the present invention and LPS, PGN, Mannose Schematic diagram.
Long oyster domain protein containing the DM9 CgDM9CP-2 of Fig. 2 present invention is to Pichia yeast inhibition schematic diagram.
Embodiment
Long oyster domain protein containing the DM9 CgDM9CP-2 of the present invention, amino acid sequence is as shown in SEQ ID NO.1.
The preparation method of above-mentioned long oyster domain protein containing DM9 CgDM9CP-2, carries out in accordance with the following steps successively：
1. the structure of recombinant vector
With specific primer P1 and P2 to long oysterCgDM9CP-2Gene coding region fragment carries out PCR amplification, the primer P1's DNA sequence dna is as shown in SEQ ID NO.2, and the DNA sequence dna of the primer P2 is as shown in SEQ ID NO.3；
PCR reaction conditions are：94 DEG C of pre-degenerations first 5 minutes, subsequently into following circulation：94 DEG C are denatured 30 seconds, and 55 DEG C are moved back Fire 30 seconds, 72 DEG C extend 45 seconds, carry out 35 circulations altogether, last 72 DEG C extend 10 minutes.Amplified fragments are purified and are recycled, with PMD19-T carriers connect.Screening positive clone after conversion, extracts plasmid；UseNde1WithXho1Two enzyme digested plasmids, use glue Recovery purifying kit（Dalian treasured bioengineering Co., Ltd）Recycling is purified to the purpose fragment that digestion produces；Recycle purpose piece Duan YujingNde1WithXho1The expression vector pET-30a connections of two enzyme digestions, complete the structure of carrier.
2. the expression of recombinant protein c gDM9CP-2
The recombinant vector translation table built is reached into host strainTransetta(DE3) in, and screening positive clone, sequencing confirm The correctness of expression cassette.Picking monoclonal, is inoculated in the LB fluid nutrient mediums of 200 mL, 220rpm, 37 DEG C of cultures to OD600 = 0.4~0.8.IPTG is added, final concentration is reached 1 mmol L-1, continue culture 4 it is small when.4 DEG C, 5000rpm, centrifuge 10 points Clock, collects thalline, is frozen in -20 DEG C spare.Take 1mL bacterium solutions to centrifuge, after supernatant discarding, add the 5 of 80 μ L water and 20 μ L Times albumen sample-loading buffer, 100 DEG C are boiled 10 minutes, are slightly centrifuged, SDS-PAGE detection expression products.
3. purifying and the renaturation of recombinant protein c gDM9CP-2
Recombinant protein uses nickel Ago-Gel FF column purifications, denaturation recombinant protein is obtained, with elution buffer dialysis renaturation. Concrete operation step is as follows：
（1）Nickel Ago-Gel FF fills column, and 1.6 × 20cm, bed volume is 10 ml；
（2）With buffer solution I（50 mmol L-1Tris-Hcl buffer solutions, pH=7.4,50 m mol L-1 NaCl, 8 mol L-1Urea）2 ~ 5 bed volumes are balanced, flow velocity is 2 ml min-1；
（3）The cell for IPTG induced expressions of learning from else's experience, is resuspended, 150 W ultrasonications 30 min, 12000 rmp, 4 with buffer solution I DEG C centrifugation 30 min, supernatant use after 0.45 μm of membrane filtration, mistake column, flow velocity is 1 ml min-1；
（4）2 ~ 5 bed volumes are washed again with buffer solution 1, and flow velocity is 2 ml min-1；
（5）With there is 50 mmol L-1The buffer solution I of imidazoles washes 2 ~ 5 bed volumes again, and flow velocity is 2 ml min-1；
（6）With 400 mmol L-1The buffer solution I elution destination proteins of imidazoles, are collected；
（7）With the expression of SDS-PAGE detection fusion albumen；
（8）5 bed volumes are washed with stream of pure water, then 3 bed volumes are washed with 20% ethanol stream, flow velocity is 2 ml min-1, column Son, which is placed in the recombinant protein for preserving in 4 DEG C of environment and being purified under denatured state, to be needed to remove urine by dialysing in renaturation buffer Element, makes albumen correctly fold again, recovers correct conformation.It is denatured reduced glutathione of the purified product through 2 mM, 0.4 mM oxygen Change what glutathione, 1 mM EDTA, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerine, 1% glycine and gradient reduced Urea dialysis renaturation, urea concentration are gradually substituted into 4 M, 3 M, 2 M, 1M, 0 M, last time dialysis to nothing from 6 M of starting Not glycerol adding during the dialyzate of urea, dialyse 12 h at 4 DEG C every time.Obtain long oyster domain protein containing DM9 CgDM9CP- 2, amino acid sequence is as shown in SEQ ID NO.1.
Length：143 amino acid
Type：Amino acid
Chain：It is single-stranded
Characteristic：Molecular weight is 16 kDa, and isoelectric point 8.64, has two conservative DM9 domains.
Experimental example 1：Long oyster domain protein containing the DM9 CgDM9CP-2 of the present invention is combined work with Mannose, LPS, PGN The detection of property
Step is as follows：
1. the 10 various PAMPs of μ g are dissolved in 50 mM sodium carbonate-bicarbonate buffer solutions respectively, the coating enzyme per 100 μ l of hole Target, 4 DEG C, overnight.
2. PBS-T is washed 3 times, 5 min, 200 μ L, 3% BSA, 37 DEG C of 1 h of closing are added per hole every time.
3. PBS-T is washed 3 times, 5 min, every hole add the long oyster of the embodiment of the present invention of 100 2 times of gradient dilutions of μ L every time Domain protein containing DM9 CgDM9CP-2,18 DEG C of 3 h of incubation.
4. PBS-T is washed 3 times, 5 min, every hole add the polyclonal antibody of the diluted anti-destination proteins of 100 μ L every time（1: 1000）, 37 DEG C of 1 h of incubation.
5. PBS-T is washed 3 times, 5 min, every hole add the goat anti rat IgG of 100 μ L alkali phosphatase enzyme marks every time（1: 4000）, 37 DEG C of 1 h of incubation.
6. PBS-T is washed 3 times, each 5min, 100 μ L are added per hole and contain 0.1% (w/v) p- nitrophenylphosphate （pNPP）With 0.5 mM MgCl250 mM sodium carbonate-bicarbonate buffer solutions（pH 9.8）, 10 ~ 30 min of lucifuge color development, 50 μ l, 2 M NaOH are added per hole and terminate color development, are read simultaneously at 405 nm of microplate reader Record numerical value.
Each hole sets 3 repetitions in experiment, to recombinate Trx albumen as protein control, during reading using TBS buffer solutions as Blank.During data analysis, experimental group（P）/ negative control group（N）>2.1 hole is considered as the positive.The results are shown in Figure 1.As a result table Bright long oyster domain protein containing the DM9 CgDM9CP-2 of the present invention has the biological activity with reference to Mannose, LPS and PGN.
Experimental example 2：Long oyster domain protein containing the DM9 CgDM9CP-2 bacteriostatic activities detection of the present invention
Comprise the following steps that：
1. the culture and preparation of microorganism
Pichia pastoris（Laboratory preserves strain）With 28 DEG C, 220 rpm of YPD culture mediums, when exponential phase is arrived in culture, use respectively Tris-HCl（50 mmol L-1, pH = 8.0）Thalline is diluted, it is about 1 × 10 to make the clump count in its every milliliter bacterium solution3CFU；
2. recombinant protein c gDM9CP-2 Antibacterial Activities
The Pichia pastoris of exponential phase uses TBS after being collected by centrifugation（50mM Tris-Hcl, 150 mM NaCl）Cleaning is resuspended （104CFU）.Long oyster domain protein containing the DM9 CgDM9CP-2 of the embodiment of the present invention of 50 μ L and isometric Pichia pastoris It is incubated at room temperature 2 h.20 μ L said mixtures are taken in flat 96 orifice plate（Costar, Fisher）In, it is separately added into 200 per hole μ L YPD and 2216E fluid nutrient mediums, 28 DEG C of shaken cultivations in microplate reader, detect and record each hole OD600 respectively every 1 h Value.Separately set rTrx protein control groups.Each sample sets three repetitions, is averaged mapping according to 3 measurement results, studies number Statistical analysis, significant difference are carried out according to using SPSS6.0 softwares（P < 0.05）Marked with *.The result is shown in Fig. 2.Experiment shows Long oyster domain protein containing the DM9 CgDM9CP-2 of the present invention can suppress the growth of Pichia pastoris.
Sequence table
<110>Dalian Ocean University
<120>Long oyster domain protein containing DM9 CgDM9CP-2, preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 143
<212> PRT
<213>Long oyster (Crassostrea gigas)
<400> 1
Met Ala Glu Trp Lys Lys Thr Ser Gly Ser Lys Ile Pro Asp Asn Ala
1 5 10 15
Ile Arg Ala Gly Tyr Glu Lys Asp Gly Lys Pro Leu Phe Ile Ala Arg
20 25 30
Ala Lys Met Gly Gly Leu Trp Thr Ser Gly Lys Cys Gly Thr His Leu
35 40 45
Pro Gly Ala His Ile Pro Tyr Asp Cys Lys Glu Leu Ile Val Lys Asp
50 55 60
Tyr Glu Val Leu Val Tyr Pro Ile Thr Ala Val Gly Phe Leu Asp Trp
65 70 75 80
Lys Gln Ala Ser Gly Gly Lys Val Pro Asp Lys Ala Phe Lys Thr Asp
85 90 95
Thr Asp Leu Tyr Val Gly Arg Ala Asn Tyr Thr Gly Ser Leu Val Pro
100 105 110
Cys Lys Ile Ser Thr Ser His Lys Cys Ala Tyr Met Gly Tyr Cys Glu
115 120 125
Lys Glu His Asn Ala Lys Glu Tyr Glu Val Leu Cys Gln Ile Lys
130 135 140
<210> 2
<211> 34
<212> DNA
<213>Artificial sequence (Crassostrea gigas)
<400> 2
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence (Crassostrea gigas)
<400> 3

Claims (3)

  1. A kind of 1. long oyster domain protein containing DM9 CgDM9CP-2, it is characterised in that：Amino acid sequence such as SEQ ID NO.1 institutes Show.
  2. A kind of 2. preparation method of long oyster domain protein containing DM9 CgDM9CP-2 as claimed in claim 1, it is characterised in that Carry out in accordance with the following steps successively：
    A. with specific primer P1 and P2 to long oysterCgDM9CP-2Gene coding region fragment carries out PCR amplification, the primer P1 DNA sequence dna as shown in SEQ ID NO.2, the DNA sequence dna of the primer P2 is as shown in SEQ ID NO.3；
    B. pcr amplification product and pET-30a carriers are passed throughNde1WithXho1Connected, converted by ligase after digestion, sequencing mirror Determine recon；
    C. above-mentioned recon is transferred to Escherichia coliTransetta（DE3）Fiber differentiation is carried out in expression bacterial strain, then purifying, Renaturation, that is, obtain the recombinant protein with amino acid sequence in sequence table SEQ ID NO.1.
  3. 3. a kind of long oyster domain protein containing DM9 CgDM9CP-2 as claimed in claim 1 is preparing suppression Pichia yeast medicine Application in thing.
CN201711418771.2A 2017-12-25 2017-12-25 Oyster containing DM9 structural domain protein CgDM9CP-2, preparation method and application Active CN108003233B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110878121A (en) * 2019-12-02 2020-03-13 大连海洋大学 CgTIMP recombinant protein capable of inhibiting toxicity of extracellular products of vibrio splendidus and preparation method thereof
CN113185593A (en) * 2021-05-18 2021-07-30 大连海洋大学 DM9 domain-containing recombinant protein rCgDM9CP-6 of crassostrea gigas, preparation method and application
CN113912691A (en) * 2021-11-01 2022-01-11 大连海洋大学 Recombinant crassostrea gigas high-mobility group protein r-CgHMGB1, preparation method and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103819550A (en) * 2014-03-03 2014-05-28 中国科学院南海海洋研究所 Novel shellfish opsonin DCP (DM9domain-containing protein) capable of promoting phagocytosis of lymphocytes

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN103819550A (en) * 2014-03-03 2014-05-28 中国科学院南海海洋研究所 Novel shellfish opsonin DCP (DM9domain-containing protein) capable of promoting phagocytosis of lymphocytes

Non-Patent Citations (3)

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Title
EMBL: "K1P333(K1P333_CRAGI)", 《UNIPROTKB》 *
SHUAI JIANG等: "DM9 Domain containing Protein Functions as a Pattern recognition receptor with Broad Microbial recognition spectrum", 《FRONTIERS IN IMMUNOLOGY》 *
刘宇: "长牡蛎含DM9结构域新型识别分子的功能研究", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110878121A (en) * 2019-12-02 2020-03-13 大连海洋大学 CgTIMP recombinant protein capable of inhibiting toxicity of extracellular products of vibrio splendidus and preparation method thereof
CN113185593A (en) * 2021-05-18 2021-07-30 大连海洋大学 DM9 domain-containing recombinant protein rCgDM9CP-6 of crassostrea gigas, preparation method and application
CN113912691A (en) * 2021-11-01 2022-01-11 大连海洋大学 Recombinant crassostrea gigas high-mobility group protein r-CgHMGB1, preparation method and application thereof
CN113912691B (en) * 2021-11-01 2023-08-11 大连海洋大学 Recombinant crassostrea gigas high mobility group protein r-CgHMGB1, preparation method and application thereof

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