CN107502612A - It is a kind of to identify with reference to WSSV laminin receptor gene C q LR and preparation method and application - Google Patents
It is a kind of to identify with reference to WSSV laminin receptor gene C q LR and preparation method and application Download PDFInfo
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- CN107502612A CN107502612A CN201710918602.9A CN201710918602A CN107502612A CN 107502612 A CN107502612 A CN 107502612A CN 201710918602 A CN201710918602 A CN 201710918602A CN 107502612 A CN107502612 A CN 107502612A
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Abstract
It is a kind of to identify with reference to WSSV laminin receptor gene C q LR and preparation method and application, it is related to white spot syndrome virus.The red claw crayfish laminin receptor is named as Cq LR, there is provided gene order, amino acid sequence, preparation method and application.Red claw crayfish Cq LR gene engineering expression products are obtained, and it is identified and combines viral activity and is identified, to the preparation for efficient anti-WSSV novel drugs.Successfully construct the Cq LR of red claw crayfish Cq LR gene engineering expression recombinant plasmid pGEX 4T 1 and corresponding prokaryotic expression system, after rCq LR recombinant expression protein sterlings are obtained, further confirm that rCq LR combine to the identifications of WSSV in vitro, that is rCq LR combinations WSSV envelope protein VP28, good basis has been established as the exploitation of anti-WSSV disease controls novel drugs for it.
Description
Technical field
The present invention relates to white spot syndrome virus (WSSV), and white spot syndrome disease is combined more particularly, to one kind identification
The red claw crayfish laminin receptor gene of malicious (WSSV) and preparation method and application.
Background technology
White spot syndrome virus (White spot syndrome virus, WSSV) is the main pathogen of prawn, additionally it is possible to
Crayfish, crab and lobster etc. are infected, white spot syndrome virus (WSSV) has turned into the major obstacle of aquatic products crustacean aquaculture, to water
The economy of production crustacean aquaculture industry causes greatly to endanger, and so far, still lacks effective drug therapy or prevention.
Laminin receptor (LR) is a kind of cell surface receptor, is referred between guided cell and cell and extracellular base
The key protein of the interphase interaction of matter, mediate the high-affinity interaction [1] between laminin and cell.LR points
For Integrins and the extended familys of nonconformity element class two.Integrins acceptor is that the signal of interest that cell and extracellular environment are linked up leads to
Road, the signal of external environment can be made a response [2] with the motion of regulating cell and migration etc..Nonconformity element family glues with layer
Even the compatibility of albumen is universal higher, and higher than integrin family, they are mostly carbohydrate-binding protein, are generally possible to identification layer adhesion
Sugar chain in protein molecular.These sugar chains are directly related with molecular recognition, signal transmission and Nasopharyngeal neoplasms isophenous [3].
Except in the function such as adhesion physiologically and connection [4], LR is also found in up-regulated expression in tumour cell, and
The invasion and attack of tumour and transfer ability is set to greatly enhance [5] in malignant tumour.In addition, there are some researches show also in tumour cell
Played an important role during angiogenesis, development and migration etc. [6].Laminin receptor can also be used as several external source venereal diseases
The cell membrane surface adsorbed receptor of substance, including prion protein [7], viral [8] and bacterium [9].For example, LR has been proved to
It is a kind of cell attachment acceptor of CSFV (CSFV), and helps CSFV to be attached to cell surface [10].Laminin
Necessary to acceptor may not be pathogenesis, but it there are and tend to and enhancing cause of disease and the ability [11] of host's interaction.
In fact, for pathogenic infection and its is pathogenic, it may be considered that block cell surface laminin acceptor and pathogen
Combination, to change the mechanism of absorption of cell in the environment.
Bibliography：
1.DiGiacomo V,Meruelo D:Looking into laminin receptor:critical
discussion regarding the non-integrin 37/67-kDa laminin receptor/RPSA
protein.Biol Rev Camb Philos Soc 2016,91(2):288-310.
2.Stipp CS:Laminin-binding integrins and their tetraspanin partners
as potential antimetastatic targets.Expert Reviews in Molecular Medicine
2010,12(15):e3.
3.Nelson J,McFerran NV,Pivato G,Chambers E,Doherty C,Steele D,Timson
DJ:The 67kDa laminin receptor:structure,function and role in disease.Biosci
Rep 2008,28(1):33-48.
4.Mckenna DJ,Simpson DA,Feeney S,Gardiner TA,Boyle C,Nelson J,Stitt
AW:Expression of the 67kDa laminin receptor(67LR)during retinal development:
correlations with angiogenesis.Experimental Eye Research 2001,73(1):81-92.
5.Givanthorwitz V,Davidson B,Reich R:Laminin-Induced Signaling in
Tumor Cells The Role of the Mr 67,000Laminin Receptor.Cancer Research 2004,64
(10):3572-3579.
6.Martin D,Zusman S,Li X,Williams EL,Khare N,Darocha S,
Chiquetehrismann R,Baumgartner S:wing blister,A New Drosophila LamininαChain
Required for Cell Adhesion and Migration during Embryonic and Imaginal
Development.Journal of Cell Biology 1999,145(1):191-201.
7.Vana K,Zuber C,Pflanz H,Kolodziejczak D,Zemora G,Bergmann AK,Weiss
S:LRP/LR as an alternative promising target in therapy of prion diseases,
Alzheimer's disease and cancer.Infect Disord Drug Targets 2009,9(1):69-80.
8.Kielian M,Chanel-Vos C,Liao M:Alphavirus Entry and Membrane
Fusion.Viruses 2010,2(4):796-825.
9.Huang SH,Jong A:Evolving role of laminin receptors in microbial
pathogenesis and therapeutics of CNS infection.Future Microbiol 2009,4(8):
959-962.
10.Gauczynski S:The 37-kDa/67-kDa laminin receptor acts as the cell-
surface receptor for the cellular prion protein.The EMBO Journal 2001,20(21):
5863-5875.
11.Simons K,Ehehalt R:Cholesterol,lipid rafts,and disease.J Clin
Invest 2002,110(5):597-603.
The content of the invention
The first object of the present invention is the gene order for providing red claw crayfish laminin receptor.
The second object of the present invention is the amino acid sequence for providing red claw crayfish laminin receptor.
The third object of the present invention is the preparation method for providing red claw crayfish laminin receptor.
The fourth object of the present invention is the application for providing red claw crayfish laminin receptor.
The gene order of the red claw crayfish laminin receptor (being named as Cq-LR) is：
The amino acid sequence of the red claw crayfish laminin receptor is：
The preparation method of the red claw crayfish laminin receptor comprises the following steps：
1) Cq-LR genes are connected in prokaryotic expression carrier pGEX-4T-1, build red claw crayfish laminin receptor
PGEX-4T-1-Cq-LR recombinant expression carriers；
2) recombination expression obtained by step 1) is carried into transformant and is directed respectively into host cell E.coli (BL21:DE3), and carry out
Induced expression, obtain expression product；
In step 2), the condition of the induced expression can be 0.1mM thiogalactosides (IPTG), inducing temperature 16
DEG C, induction time 20h.
3) purification procedures 2) obtained expression product, pass through the recombinant protein that affinity chromatography obtains higher degree
CqLR-GST。
The Cq-LR can identify shrimp white spot syndrome virus (WSSV) and have dose dependent, therefore it is being probed into
How WSSV, which infects entering for shrimps cell, has researching value in born of the same parents' mechanism, to prepare anti-WSSV kind new medicines and animal disease resistant feed
Additive lays the first stone.
The application of the red claw crayfish laminin receptor includes the structure of expression vector, and it is in host cell
Expression, and expression product isolate and purify and virus identification activity analysis, gene engineering product can identify simultaneously in vitro
It is a kind of genetic engineered product for having and identifying and combining WSSV activity, for as poisoning intrusion cell target spot with reference to WSSV
Designing anti-guttate morphea syndrome disease active drug preventing and treating has important application prospect.
The present invention successfully builds recombination expression on the basis of isolated rCq-LR according to Cq-LR gene sequence characteristics
Carrier is simultaneously expressed in E. coli system and purifies acquisition restructuring rCq-LR albumen, and the recombinant protein can identify that prawn white spot is comprehensive
Simulator sickness virus (WSSV) simultaneously combines WSSV envelope proteins VP28.Result of study shows that Cq-LR may take part in WSSV and infect red chela
Crayfish enters born of the same parents' process, and therefore, recombination engineering product Cq-LR enters born of the same parents' mechanism probe into how WSSV to infect shrimps cell
In there is reliable researching value, and show very tempting application prospect in anti-WSSV kind new medicines development and application are prepared.
Brief description of the drawings
Fig. 1 is pGEX-4T-1-Cq-LR Prokaryotic expression vector construction figures.
Fig. 2 is the electrophoresis pattern that SDS-PAGE analyzes pGEX-4T-1-Cq-LR recombinant vector clone induced expressions.M is
SDS-PAGE standard proteins Marker, 1 is the bacterium solution supernatant that empty carrier imports expression bacterium, and 2 be the expression for importing recombinant vector
Bacterium solution supernatant before bacterium addition derivant, 2 be the bacterium solution supernatant after the expression bacterium addition derivant of importing recombinant vector, it is seen that about
70kDa induction expression protein band.
Fig. 3 is the electrophoresis pattern that SDS-PAGE analyzes the purifying of pGEX-4T-1-Cq-LR recombinant vector clones expression product.
M is SDS-PAGE standard proteins Marker, and 1 is the Cq-LR recombinant proteins of expression, it is seen that about 70kDa obvious albumen one
Band.
Fig. 4 is that Pull down experimental verifications rCq-LR can be identified with reference to the main cyst membrane Viral structural protein VP2s 8 of WSSV.
Embodiment
Technical scheme is described with reference to the accompanying drawings by the following examples.
The structure of the red claw crayfish laminin receptor Cq-LR prokaryotic expression carriers of embodiment 1
Amplification coding Cq-LR (cDNA) genes ORF specific forward primer F1 and anti-sense primer R1 is designed, is drawn in upstream
Thing F 5 ' end addition EcoR I restriction enzyme sites；In anti-sense primer R 5 ' end addition Sal I restriction enzyme sites, and its ORF is carried out
PCR is expanded.
Sense primer F：5 '-CCGGAATTCATGTCGGGAGGACTTGCTGTTATGA-3 ', anti-sense primer R：5′-
ACGCGTCGACTTACCAGTTGGTACCATCACCTGCA-3′。
PCR reaction conditions are：98 DEG C of pre-degeneration 3min；98 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 60s, are repeated
30 circulations；72 DEG C of extension 10min.
PCR primer is reclaimed using agarose gel purification kit, the PCR primer of recovery, then PCR primer is carried out
EcoRI/Sal I double digestions, and be connected with EcoR I/Sal I double digestions linearisation prokaryotic expression carrier pGEX-4T-1, obtain
Recombinant expression carrier pGEX-4T-1-Cq-LR.Sequencing identification reading frame is accurate.
PGEX-4T-1-Cq-LR vector construction figures are referring to Fig. 1.
Recombinant expression carrier pGEX-4T-1-Cq-LR is in E. coli (BL21 for embodiment 2:DE3 the induction in)
Expression
Induced expression：
1st, the recombinant expression carrier Transformed E .coli BL21 that will be built, and with IPTG induced expressions.
2nd, picking monoclonal bacterium colony, it is inoculated in 5ml and contains Amp+LB culture mediums in, 37 DEG C, 200rpm shaking table cultures 12h.
3rd, 20ml is seeded to 1 ︰ 100 ratio and contains Amp+LB culture mediums in, 37 DEG C, 200rpm shaking table cultures to OD600
For 0.3 to 0.5.
4th, IPTG to final concentration 0.1mM is added, 150rpm induces 20h at 16 DEG C.
As a result show, see Fig. 2, can detect obvious induction band after IPTG inductions in thalline, size is 70kDa left
The right side, illustrate that the condition can obtain the expression product of higher proportion.
Purifying：
1st, thalline is collected, is resuspended in PBS, ultrasonication, and in 4 DEG C, 12000rpm centrifuges 5min.
2nd, as above thalline ultrasonication supernatant can centrifuge 10min again, fully to remove not lysosome agglomerate, and with 0.22
μm filter filtering, you can carry out affinitive layer purification.
3rd, chromatographic column is cleaned with 5~10 column volume MilliQ water.
4th, balance：Chromatographic column is balanced with 5~10 column volume PBSs.
5th, hanging column：Soluble upper after filtering is all crossed into post with 0.8ml/min.
6th, post is washed：Post is crossed with the PBS of 5~10 column volumes, washes away non-specific binding albumen.
7th, elute：Utilize eluent (50mM Tris-HCl [pH7.4], 500mM NaCl, 20mM reduced glutathione)
Eluted.
8th, collect：Elution albumen is collected, takes a small amount of progress SDS-PAGE electroresis appraisals.
As a result show, the corresponding more single protein band (Fig. 3) of purifying eluting peak, illustrating can after affinitive layer purification
To obtain the higher recombinant expression protein of purity.
Embodiment 4 proves that Cq-LR recombinant proteins are combined with WSSV identification using Pull down experiments
Probe into whether rCqLR can interact with WSSV (VP19, VP24, VP26 and VP28) main envelope protein.
4 kinds of VPs ORFs (ORF) are expanded by gene-specific primer (table 1) first, and are cloned into PB513B-Flag carriers
In.Transfected for DNA, human embryo kidney (HEK) 293T (HEK 293T) is inoculated with and trained in 37 DEG C of Dulbecco's ModifiedEagle
Support in base (Thermo Fisher) and grow.PB513B-VPs-Flag plasmids are transfected into HEK293T respectively with transfection reagent.
After transfecting 72h, with 4 DEG C of cell lysis 2h of Western and IP cell lysis buffer solutions.After 500g centrifugations 5min, to every kind of supernatant
Liquid is divided into two, and is separately added into 20 μ g CqLR-GST albumen and GST protein, and all samples all add 20 μ L glutathione
Sepharose 4B resin bead, and it is incubated 2h in 4 ° of rotations.After incubation, resin bead is washed 5 times with PBS, with 20 2 × SDS- of μ L of addition
PAGE sample buffers (100mM Tris, 4%SDS, 20% glycerine, 2% beta -mercaptoethanol, 0.2% bromophenol blue, pH6.8) boil
Boil 10min denaturation.By protein example, simultaneously electricity goes to pvdf membrane (GE Healthcare) to electrophoresis in 15%SDS-PAGE gels
On.Film is placed in 5% degreasing of TBST buffer solutions (20mM Tris, 150mM NaCl, 0.1%Tween 20, pH 7.6) dissolving
Room temperature closes 1h in milk powder, is then stayed overnight with anti-flag monoclonal antibodies (Sigma, 1 ︰ 10000) at 4 DEG C.Then TBST is used
Buffer solution washing film 5 times, the antibody (1 ︰ 5000) then marked at room temperature with horseradish peroxidase (HRP) is incubated 1h.Most
Developed afterwards using horseradish peroxidase HRP-ECL luminescence methods.As a result such as Fig. 4, it was demonstrated that rCq-LR can identify WSSV and combine
WSSV envelope proteins VP28.
It is contemplated that obtaining red claw crayfish Cq-LR gene engineering expression products, and it is identified and combines the work of virus
Property identified, to the preparation for efficient anti-WSSV novel drugs.The present invention successfully constructs red claw crayfish Cq-LR genes
Engineering recombinant expression pGEX-4T-1-Cq-LR and corresponding prokaryotic expression system, obtaining, rCq-LR recombinant expression proteins are pure
After product, further confirm that rCq-LR combines to the identifications of WSSV in vitro, be i.e. rCq-LR combinations WSSV envelope protein VP28, be
It has established good basis as the exploitation of anti-WSSV disease controls novel drugs.
Sequence table
<110>Xiamen University
<120>It is a kind of to identify with reference to WSSV laminin receptor gene C q-LR and preparation method and application
<141> 2017-09-30
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1000
<212> DNA
<213>Red claw crayfish (Cherax quadricarinatus)
<400> 2
acgcgggctt cttcttgatg tgatcctgcc gaggagcgga tagccgccat catgtcggga 60
ggacttgctg ttatgagcct tgaggagaat gatgtgacaa ggtttcttgc agcatcaact 120
catttgggtg ccaacaatgc aaactttcag atggaacaat acgtcttcaa gaggcgtcag 180
gatggtgttc acatcatcca tttgcgcaag acctatgaga agatcctgct agcagcacgt 240
gcaattgctg ccattgaaaa tcctgctgat gtgtatgtga tatcatcacg ccccatggga 300
cagagagctg tactcaaatt tgcaagatac actggtgcca ctccaattgc tgggcgcttc 360
actcctggag cattcaccaa ccaaatccag gctgctttcc gtgaacctcg actgttagtt 420
gtgactgacc ctgcttcgga ccatcagccc ataactgaag catcttatgt taatatccct 480
gttattggat tttgcaacac tgattctccc cttcgttttg tggacgttgc tatcccatgt 540
aataacaaga gtcctcactc agttggtctg atttggtgga tgttggctcg tgaagttcta 600
cgtctgcgtg gcaccatttc ccgcaacctt ccttgggaga ctgacgttat gcctgatttg 660
ttcttctacc gtgaccctga ggaacaggag aaggaagaag ctgccaaagc tgaggctgcc 720
aaggctgaag cagaggcagc taaggcagaa gttccagccc cagagacttg ggtcaatgat 780
gtagctgatg cagaggctcc tgttgctgca cctgccacac ctgttgctgc tagtacagct 840
ccagtggctg gtgtgggcat accacctgct gctcctgcaa ctgttgatga ctggggtcaa 900
gctggtgatg actgggctgc tcctgctgct ggaactggcg atgattgggg tggtgcaggt 960
gatggtacca actggtaaac ctcaataaat aatagtcggc 1000
<210> 2
<211> 308
<212> PRT
<213>Red claw crayfish (Cherax quadricarinatus)
<400> 2
Met Ser Gly Gly Leu Ala Val Met Ser Leu Glu Glu Asn Asp Val Thr
1 5 10 15
Arg Phe Leu Ala Ala Ser Thr His Leu Gly Ala Asn Asn Ala Asn Phe
20 25 30
Gln Met Glu Gln Tyr Val Phe Lys Arg Arg Gln Asp Gly Val His Ile
35 40 45
Ile His Leu Arg Lys Thr Tyr Glu Lys Ile Leu Leu Ala Ala Arg Ala
50 55 60
Ile Ala Ala Ile Glu Asn Pro Ala Asp Val Tyr Val Ile Ser Ser Arg
65 70 75 80
Pro Met Gly Gln Arg Ala Val Leu Lys Phe Ala Arg Tyr Thr Gly Ala
85 90 95
Thr Pro Ile Ala Gly Arg Phe Thr Pro Gly Ala Phe Thr Asn Gln Ile
100 105 110
Gln Ala Ala Phe Arg Glu Pro Arg Leu Leu Val Val Thr Asp Pro Ala
115 120 125
Ser Asp His Gln Pro Ile Thr Glu Ala Ser Tyr Val Asn Ile Pro Val
130 135 140
Ile Gly Phe Cys Asn Thr Asp Ser Pro Leu Arg Phe Val Asp Val Ala
145 150 155 160
Ile Pro Cys Asn Asn Lys Ser Pro His Ser Val Gly Leu Ile Trp Trp
165 170 175
Met Leu Ala Arg Glu Val Leu Arg Leu Arg Gly Thr Ile Ser Arg Asn
180 185 190
Leu Pro Trp Glu Thr Asp Val Met Pro Asp Leu Phe Phe Tyr Arg Asp
195 200 205
Pro Glu Glu Gln Glu Lys Glu Glu Ala Ala Lys Ala Glu Ala Ala Lys
210 215 220
Ala Glu Ala Glu Ala Ala Lys Ala Glu Val Pro Ala Pro Glu Thr Trp
225 230 235 240
Val Asn Asp Val Ala Asp Ala Glu Ala Pro Val Ala Ala Pro Ala Thr
245 250 255
Pro Val Ala Ala Ser Thr Ala Pro Val Ala Gly Val Gly Ile Pro Pro
260 265 270
Ala Ala Pro Ala Thr Val Asp Asp Trp Gly Gln Ala Gly Asp Asp Trp
275 280 285
Ala Ala Pro Ala Ala Gly Thr Gly Asp Asp Trp Gly Gly Ala Gly Asp
290 295 300
Gly Thr Asn Trp
305
Claims (7)
1. the gene order of red claw crayfish laminin receptor is：
2. the amino acid sequence of red claw crayfish laminin receptor is：
3. the preparation method of red claw crayfish laminin receptor, it is characterised in that comprise the following steps：
1) Cq-LR genes are connected in prokaryotic expression carrier pGEX-4T-1, build red claw crayfish laminin receptor
PGEX-4T-1-Cq-LR recombinant expression carriers；
2) recombination expression obtained by step 1) is carried into transformant and is directed respectively into host cell E.coli (BL21:DE3), and induced
Expression, obtains expression product；
3) purification procedures 2) obtained expression product, pass through the recombinant protein c qLR- that affinity chromatography obtains higher degree
GST。
4. the preparation method of red claw crayfish laminin receptor as claimed in claim 3, it is characterised in that in step 2), institute
The condition for stating induced expression is 0.1mM thiogalactosides, 16 DEG C of inducing temperature, induction time 20h.
5. the application of red claw crayfish laminin receptor, it is characterised in that the Cq-LR identifies shrimp white spot syndrome virus.
6. application as claimed in claim 5, it is characterised in that the shrimp white spot syndrome virus infection shrimps cell enters born of the same parents
Mechanism, prepare anti-WSSV kind new medicines and animal disease resistant feed addictive.
7. application as claimed in claim 5, it is characterised in that the application of the red claw crayfish laminin receptor includes gene
The structure of expression vector, its expression in host cell, and expression product isolate and purify and virus identification activity analysis.
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| CN107903319A (en) * | 2017-12-28 | 2018-04-13 | 厦门大学 | Expression and application of a kind of thymosin extrasin PROTEIN C q TRP1 of Anti-infection to WSSV in Pichia pastoris |
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| CN108558993A (en) * | 2018-01-12 | 2018-09-21 | 厦门大学 | It is a kind of inhibit WSSV infection Cq-Ns1abp genes and its albumen antiviral activity application |
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| CN114574526A (en) * | 2021-01-28 | 2022-06-03 | 江苏集萃药康生物科技股份有限公司 | Construction method of RPSA gene humanized mouse model |
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Cited By (7)
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| CN107903319A (en) * | 2017-12-28 | 2018-04-13 | 厦门大学 | Expression and application of a kind of thymosin extrasin PROTEIN C q TRP1 of Anti-infection to WSSV in Pichia pastoris |
| CN108558993A (en) * | 2018-01-12 | 2018-09-21 | 厦门大学 | It is a kind of inhibit WSSV infection Cq-Ns1abp genes and its albumen antiviral activity application |
| CN108558993B (en) * | 2018-01-12 | 2021-06-11 | 厦门大学 | Cq-Ns1abp gene for inhibiting WSSV infection and application of protein antiviral activity of Cq-Ns1abp gene |
| CN108285481A (en) * | 2018-01-30 | 2018-07-17 | 厦门大学 | The gene C q-BAF and preparation method of promotion WSSV infection and application |
| CN109913485A (en) * | 2019-03-25 | 2019-06-21 | 南京农业大学 | A method of it prepares, purify HOPS complex proteins |
| CN114574526A (en) * | 2021-01-28 | 2022-06-03 | 江苏集萃药康生物科技股份有限公司 | Construction method of RPSA gene humanized mouse model |
| CN114574526B (en) * | 2021-01-28 | 2024-03-12 | 江苏集萃药康生物科技股份有限公司 | Construction method of RPSA gene pig-derived mouse model |
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Application publication date: 20171222 |