CN108018289A - A kind of gene C q-VCP of suppression WSSV early infections and its preparation and application - Google Patents
A kind of gene C q-VCP of suppression WSSV early infections and its preparation and application Download PDFInfo
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Abstract
A kind of gene C q VCP of suppression WSSV early infections and its preparation and application, are related to white spot syndrome virus.The red claw crayfish valosin-containing protein is named as Cq VCP.Cq VCP genes are connected to carrier pMD18 T, and analyze its open reading frame, obtain Cq VCP gene orders；The Cq VCP gene orders of gained carry out phylogenetic analysis and analyze its protein structure domain.The red claw crayfish valosin-containing protein can be applied in Anti-infection to WSSV medicine is prepared.It is related to new gene and its protein function, i.e. red claw crayfish VCP gene magnifications and its functional verification.During WSSV infects host cell, the early stage that Cq VCP can regulate and control virus enters born of the same parents and subsequent Vesicle transport process.Cq VCP regulate and control the WSSV early infection stages, for having important application prospect as the anti-guttate morphea syndrome disease active drug prevention of poisoning intrusion cell shot design.
Description
Technical field
The present invention relates to white spot syndrome virus, enters born of the same parents and born of the same parents to it more particularly, to host cell in WSSV courses of infection
Interior transport process plays gene C q-VCP and its preparation and application of a kind of suppression WSSV early infections of regulating and controlling effect.
Background technology
White spot syndrome virus (White spot syndrome virus, WSSV) is the important crust such as prawn and crayfish
One of most destructive viral cause of disease of class aquaculture, can cause extensive guttate morphea syndrome disease to break out and be infected dynamic
The high mortality of thing, causes greatly to endanger to the economy of aquatic products crustacean aquaculture industry, so far, still lacks effective
Drug therapy or preventive means.
Valosin-containing protein (valosin-containing protein, VCP) is highly conserved adenosinetriphosphataes
A member in superfamily (ATPases associated with various cellular activities, AAA)[1], ginseng
With various kinds of cell activity regulation, including protein degradation, film fusion, anti-apoptotic, the endoplasmic reticulum phase that Ubiquitin-proteasome relies on
Close degraded and nucleic acid reparation etc.[2].The realization of the various biological functions of VCP in the cell depends in connection each
Kind co-factor and the adaptor protein with its interaction[3].The co-factor combined with VCP is broadly divided into substrate and raises co-factor and bottom
Thing processes co-factor, the former includes Ufd1-Npl4 heterodimers, Shp1/p47 and Derlin-1 etc.；The latter mainly includes
VCIP135 and SVIP etc..
WSSV is double-stranded DNA virus, and the course of infection of DNA virus, is generally divided into and sticks, enter born of the same parents, intracellular transport, viral core
Acid enters core and transcription duplication, progeny virus assembling and maturation[4].In the endocytic pathway of viral early stage invasion host cell,
VCP participates in clathrin-mediated endocytosis process by connecting clathrin clathrin and adaptor protein Aps[5], grind before
Study carefully report and have verified that clathrin-mediated endocytosis process is exactly the crucial main path of WSSV infection host cells[6].In intracellular
In inner body transport process, VCP is combined with co-factor UBXD1, promotes the albumen of ubiquitination to sort and degrade.And VCP dysfunctions
Late endosomal can be caused to assemble, influence intraluminal substrate sorting, and subsequent vesica is degraded with lysosome fusion[7].Coronal
In viral early infection, Drug inhibition VCP functions, the endosomal maturation that can suppress transhipment virus discharges so as to suppress it
Come[8].The mosquito-borne sindbis alphavirus in storehouse (Sindbis virus) can infect drosophila and mammalian cell.Clpp gene drop or
Drug inhibition VCP functions, can influence virus and enter born of the same parents in early days and change the Vesicle transport approach of virus receptor[9].In addition VCP exists
poliovirus[10]、hepatitis B virus[11]Course of infection Deng virus also plays certain effect.
Bibliography：
1.Erzberger J P,Berger J M.Evolutionary relationships and structural
mechanisms of AAA+proteins[J].Annual Review of Biophysics&Biomolecular
Structure,2006,35(1):93.
2.Meyer H,Bug M,Bremer S.Emerging functions of the VCP/p97AAA-ATPase
in the ubiquitin system.[J].Nature Cell Biology,2012,14(2):117.
3.Yeung H O,Kloppsteck P,Niwa H,et al.Insights into adaptor binding
to the AAA protein p97.[J].Biochemical Society Transactions,2008,36(1):62-7.
4.Marsh M,Helenius A.Virus entry:open sesame[J].Cell,2006,124(4):729.
5.Pleasure I T,Black M M,Keen J H.Valosin-containing protein,VCP,is a
ubiquitous clathrin-binding protein[J].Nature,1993,365(6445):459-462.
6.Chen R,Shen K,Zhen C,et al.White spot syndrome virus entry is
dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP
in a CME-dependent manner[J].Scientific Reports,2016,6:28694.
7.Ritz D,Vuk M,Kirchner P,et al.Endolysosomal sorting of
ubiquitylated caveolin-1 is regulated by VCP and UBXD1and impaired by VCP
disease mutations.[J].Nature Cell Biology,2011,13(9):1116-23.
8.Wong H H,Kumar P,Tay F P,et al.Genome-Wide Screen Reveals Valosin-
Containing Protein Requirement for Coronavirus Exit from Endosomes.[J]
.Journal of Virology,2015,89(21):11116.
9.Panda D,Rose P P,Hanna S L,et al.Genome-wide RNAi screen identifies
SEC61A and VCP as conserved regulators of Sindbis virus entry[J].Cell
Reports,2013,5(6):1737-48.
10.Arita M,Wakita T,Shimizu H.Valosin-containing protein(VCP/p97)is
required for poliovirus replication and is involved in cellular protein
secretion pathway in poliovirus infection[J].J Virol,2012,86(10):5541-5553.
11.Jiao BY,Lin WS,She FF,et al.Hepatitis B virus X protein enhances
activation of nuclear factor kappaB through interaction with valosin-
containing protein[J].Arch Virol,2011,156(11):2015-2021.
The content of the invention
The first object of the present invention is the gene order for providing red claw crayfish valosin-containing protein.
The second object of the present invention is to provide red claw crayfish valosin-containing protein amino acid sequence.
The third object of the present invention is the preparation method for providing red claw crayfish valosin-containing protein.
The fourth object of the present invention in provide red claw crayfish valosin-containing protein in Anti-infection to WSSV medicine is prepared
Using.
The red claw crayfish valosin-containing protein (valosin-containing protein) is named as Cq-VCP.
The gene order of the red claw crayfish valosin-containing protein is：
The red claw crayfish valosin-containing protein amino acid sequence is：
The preparation method of the red claw crayfish valosin-containing protein comprises the following steps：
1) Cq-VCP genes are connected to carrier pMD18-T, and analyze its open reading frame (ORF), obtain Cq-VCP genes
Sequence；
2) the Cq-VCP gene orders obtained by step 1), carry out phylogenetic analysis and analyze its protein structure domain.
The Cq-VCP is adenosinetriphosphataes superfamily (ATPases associated with various
Cellular activities, AAA) in a member, it has the conservative of height with typical domain.
The red claw crayfish valosin-containing protein can be applied in Anti-infection to WSSV medicine is prepared.
The present invention relates to a kind of new gene and its protein function, i.e. red claw crayfish (Cherax quadricarinatus)
VCP gene magnifications and its functional verification.During WSSV infects host cell, the early stage that Cq-VCP can regulate and control virus enters born of the same parents
And subsequent Vesicle transport process.Cq-VCP regulates and controls the WSSV early infection stages, for as poisoning intrusion cell shot design
Anti- guttate morphea syndrome disease active drug prevention has important application prospect.The present invention on the basis of amplification obtains Cq-VCP,
Its expression is disturbed according to Cq-VCP gene orders design dsRNA is obtained, and suppresses its function using its specific drugs DBeQ.Research
The result shows that Cq-VCP function inhibitios, can reduce viropexis and virus replication.Therefore, Cq-VCP is probing into WSSV such as
There is important research value in the viral intracellular transport mechanism of what infection shrimps cell, and it is anti-being prepared using VCP as target spot
There is good potential application prospect in WSSV infection new drug development applications.
Brief description of the drawings
Fig. 1 predicts for Cq-VCP domains.In Fig. 1, Cq-VCP is by CDC48_N, CDC48_2 and 2 AAA domains
(commonly referred to as D1, D2) totally 4 typical domains are formed, the function of wherein D1 and D2 performance ATPase enzymes.
Fig. 2 is that WSSV infects reduction after striking drop cell Inner source VCP using double-stranded RNA perturbation technique.In fig. 2, WSSV,
MOI=10, infection cell 1h, then detection find that WSSV enters born of the same parents and measures reduction.
Fig. 3 is that WSSV replicates reduction after striking drop cellular endogenous VCP using double-stranded RNA perturbation technique.In figure 3, MOI=1,
WSSV infects 3h, 6h, 12h, it is found that its early gene IE1 and late gene VP28 is replicated significantly reduces.
Fig. 4 suppresses WSSV for VCP specific drugs DBeQ to be replicated.In Fig. 4, DBeQ medicines significantly inhibit WSSV genes IE1
With the duplication of VP28, low concentration (5 μm/L) effect is less than high concentration (15 μm/L), show its for virus infection have concentration according to
Lai Xing.
Fig. 5 suppresses WSSV degradeds for VCP specific drugs DBeQ.In Figure 5, high concentration (15 μm/L) DBeQ medicines significantly press down
The duplication of WSSV genes IE1 and VP28 processed, and the degraded of togavirus is significantly suppress, show melting which inhibits virus envelope
Close and occur and degrade.
Embodiment
Technical scheme is described with reference to the accompanying drawings by the following examples.
1 red claw crayfish valosin-containing protein Cq-VCP gene clonings of embodiment
Extraction red claw crayfish Hpt cell RNAs, template of the part through RT-PCR reverse transcriptions into cDNA as gene magnification,
Another part synthesizes the first chains of cDNA of 3' and 5'RACE.According to the red claw crayfish hematopoietic tissue transcript profile of laboratory structure early period
In part Cq-VCP gene orders design specific gene amplimer expanded.
Sense primer F：5′-ATGGCCGAACAGGAAGACTTAGC-3′；
Anti-sense primer R：5′-CAAACCACTCATGAATGGTACACTAAT-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 90s, repeat
30 circulations；72 DEG C of extension 10min.
PCR product, the PCR product of recycling, through connecting with pMD18-T carriers are recycled using agarose gel purification kit
Connect, convert to bacillus coli DH 5 alpha, analysis is compared after sequencing to determine gained gene order as Cherax
VCP genes, that is, Cq-VCP of quadricarinatus.
Fig. 1 is shown in the prediction of Cq-VCP domains.
Embodiment 2Cq-VCP clpp genes drop enters WSSV infection Hpt cells the influence of born of the same parents
Cq-VCP double-stranded RNAs (dsRNA) are synthesized according to table 1 to be used to disturb.Culture red claw crayfish hpt cells are prepared, are added
(10 are cultivated in 96 ghost culture plates5Cells/ holes).Then 1 μ g dsRNA, 0.8 μ L cellfectin and 14 μ L DEPC are taken
Water is incubated at room temperature 15min after flicking mixing, and benefit L15 culture mediums to 50 μ L add to 96 porocyte culture plates per hole in culture cell,
1/2 culture medium is replaced after 24h, dsRNA is added again with reference to the above method, dsRNA used in control is dsGFP RNA.Gene is done
Disturb and WSSV is infected with MOI=10 after 36h, infect and collect cell sample with 1 × SDS cracking after 1h, Western blot detections are sick
Poison enters the change of born of the same parents' amount.
Table 1
The result is shown in Fig. 2, WSSV infection enter born of the same parents during, relative to control group, after Cq-VCP clpp genes drop, Cq-VCP
Expressing quantity reduces, so as to cause WSSV envelope proteins VP28 reductions, shows that WSSV enters born of the same parents and measures reduction.
Embodiment 3Cq-VCP strikes drop and transcribes what is replicated afterwards to WSSV infection red claw crayfish hematopoietic tissue cells (Hpt cells)
Influence
Cq-VCP genes dsRNA is synthesized according to table 1 to be used to disturb.Culture red claw crayfish hpt cells are prepared, it is empty thin to add 24
(5 × 10 are cultivated in born of the same parents' culture plate5Cells/ holes).1 μ g dsRNA, 0.8 μ L cellfectin is taken to be flicked with 14 μ L DEPC water
15min is incubated at room temperature after mixing, L15 culture mediums is mended and is added to 100 μ L in hole, 1/2 culture medium is changed after 24h, according to the above method again
Secondary addition dsRNA, dsRNA used in control are dsGFP.WSSV is infected with MOI=1 after gene interference 36h, infects 3h, 6h, 12h
Cell sample is collected in cracking afterwards, is extracted RNA, is used the PrimeScriptTM RT reagentKit reverse transcriptions of Takara companies
Synthesize cDNA, the expression of semiquantitive PCR detection VCP genes jamming effectiveness, viral pole early gene IE1 and late gene VP28
Amount, situation is replicated with the transcription of clear and definite virus.
The result is shown in Fig. 3,3h, 6h, 12h after WSSV infection, relative to control group, after Cq-VCP clpp genes drop, Cq-
VCPmRNA is horizontal to be reduced, so as to cause the expression quantity of WSSV poles early gene IE1 and late gene VP28 to reduce, shows WSSV
Transcription replication capacity weaken.
Embodiment 4VCP inhibitor DBeQ transcribes the influence of duplication after infecting WSSV Hpt cells
Selection is directed to VCP-ATPase activity specific drugs, and L15 culture mediums are diluted to 5 μm of working concentration/L and 15 μm/L,
Pretreatment cell 4h, then with MOI=1, after WSSV infection cells 6,12h cracking collect cell sample.Semiquantitive PCR detects
The expression quantity of VCP genes jamming effectiveness, viral pole early gene IE1 and late gene VP28, is replicated with the transcription of clear and definite virus
Situation.Suppress the duplication of virus using cycloheximide (CHX, 100 μ g/mL) at the same time, then Western blot detections virus drop
The change of solution.
The result is shown in Fig. 4 and 5, infects 6h to 12h in WSSV, 15 μm/L of high concentration significantly reduces late genes
VP28mRMA, and 5 μm/L of low concentration does not influence, and illustrates that DBeQ can significantly inhibit the duplication of virus；It is complete using cycloheximide CHX
The full duplication for suppressing virus, WSSV infection 6h to 12h, causes relative to DBeQ5 μm/L of low concentration, DBeQ15 μm of high concentration/L
WSSV envelope proteins VP28 is raised, and illustrates that DBeQ suppresses virus degraded；DBeQ15 μm of high concentration/L processing cells, WSSV infection 6h
To 12h, WSSV late genes VP28mRNA is horizontal to be reduced, while envelope protein VP28 is significantly reduced, and is illustrated as the time becomes
Change DBeQ and significantly inhibit WSSV duplications.
Sequence table
<110>Xiamen University
<120>A kind of gene C q-VCP of suppression WSSV early infections and its preparation and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 2385
<212> DNA
<213> Cherax quadricarinatus
<400> 2
atggccgaac aggaagactt agcaacagct atactgaaag agaagaaaaa gccaaaccgc 60
ctcatcgtgg aagatgcagt caatgatgac aattctgttg tggcactcag ccaggctaag 120
atggatgagc tgcagctctt tcgaggtgac acagtgctca tcaagggcaa gaaacgcaag 180
cagactgtct gcattgtgct ctctgatgac accatgtctg atgacaaagt tcgcatgaat 240
cgtgtagtga gaaacaatct tcgcattcgt ttgggtgatg tggtagctat ccagccctgc 300
ccagacgtga agtatggcaa gcgtatacat gttctaccta ttgatgacac agttgaaggt 360
ctgactggta atatttttga ggtatttctg aaaccgtact ttctggaagc atatcgaccc 420
atccacaaag gtgacctctt tctcgtccgt ggaggtatga gagctgtaga gttcaaagta 480
gtggagacag acccagcccc ttactgcatc gtatcccagg acactgttat ttactgcgaa 540
ggggaaccag tgaagcgaga agaagaagag gaacagttga atgaagtggg ctatgatgat 600
attggtgggt gcaggaagca gttagcacaa atcaaagaga tggtagaatt gccattgcgc 660
catccttctc tattcaaagc cattggtgtc aagccaccaa gaggtatatt attgtatggc 720
ccaccaggca caggaaaaac actcattgca cgagctgttg cgaatgagac tggagcattc 780
ttctttttga taaatggccc agaaattatg tctaaattag caggtgaatc tgaaagtaat 840
cttcggaaag catttgaaga ggcagaaaaa aattccccag ccataatttt catagacgag 900
attgatgcca ttgcaccaaa acgtgaaaag acacatggag aagttgaacg acgaatagta 960
tcacagctat tgacactgat ggatggcttg aagcagcgct ctcatgttat tgttatggct 1020
gccacaaaca ggcctaactc tattgatcct gctcttaggc gatttggtcg ttttgaccga 1080
gaagttgaca tcagtattcc agacaccaca ggtcgcctgg aagtcctgcg aatccacact 1140
aagaacatga aattgtccga tgatgttgac ctagaacaga ttgctgcaga gactcatggc 1200
catgttggtg cagatttggc tgctctgtgc tcagaagctg cccttcaaca aatcagagaa 1260
aaaatggatc ttattgatct tgatgatgac cagattgatg ctgaggtact taattcacta 1320
gcagtaacta tggagaactt caggtttgcc atgggtaaga gcacaccatc agccctccgt 1380
gaaacagtag tggaggtgcc caacattacc tggcatgata ttggtggctt agaaaatgtc 1440
aagagagagt tacaggagct tgtccagtat cctgtggagc acccagacaa attcctgaag 1500
tttggcatga ctcccagcaa gggggtacta ttctatggcc ctcctggttg tggtaaaacc 1560
ctgttagcaa aggctattgc taatgagtgt caagctaact tcatctccat caagggtcca 1620
gagctgctta ccatgtggtt tggtgaatca gaagctaatg tgagagatgt ctttgataag 1680
gctcgtgcag cagctccgtg tgtcttgttc tttgacgagt tggacagcat tgccaaggct 1740
cgaggtggct ctgcaggtga tgcaggtggt gctgctgatc gagtcataaa tcaggtacta 1800
acagaaatgg atggtatggg agccaaaaag aatgtcttca tcattggtgc aacaaaccga 1860
cctgatatta ttgatccagc cattttacga cctgggcgtc tggaccagtt gatctacata 1920
cctctccccg atgaaaagtc tagagtgcag atcctgaagg cgtgcttgag gaaatcccca 1980
gtgtcaaaga gagttgatct ggaatatctt gccaaagtgt cccatggatt ctctggtgct 2040
gatttaacag aaatttgcca gagggcttgc aaactggcaa ttcggcaggc tattgaagca 2100
gacataaagc gggagagaga gagagctgct ggagacaata tggatatgga ggaagaggat 2160
cctgtaccag agataactcg ggatcacttt gaggaagcta tgaagtatgc ccgccgttca 2220
gtctctgaca acgacattcg caaatatgag atgttctccc agacgctgca gcagagtcga 2280
gggtttggct ctaatttcag atttccagac cagcaaggcc agggaggcag cagccatggt 2340
ggaaactttg gtgcagatgg agaggatgat gatttgtact cgtaa 2385
<210> 2
<211> 794
<212> PRT
<213>Red claw crayfish (Cherax quadricarinatus)
<400> 2
Met Ala Glu Gln Glu Asp Leu Ala Thr Ala Ile Leu Lys Glu Lys Lys
1 5 10 15
Lys Pro Asn Arg Leu Ile Val Glu Asp Ala Val Asn Asp Asp Asn Ser
20 25 30
Val Val Ala Leu Ser Gln Ala Lys Met Asp Glu Leu Gln Leu Phe Arg
35 40 45
Gly Asp Thr Val Leu Ile Lys Gly Lys Lys Arg Lys Gln Thr Val Cys
50 55 60
Ile Val Leu Ser Asp Asp Thr Met Ser Asp Asp Lys Val Arg Met Asn
65 70 75 80
Arg Val Val Arg Asn Asn Leu Arg Ile Arg Leu Gly Asp Val Val Ala
85 90 95
Ile Gln Pro Cys Pro Asp Val Lys Tyr Gly Lys Arg Ile His Val Leu
100 105 110
Pro Ile Asp Asp Thr Val Glu Gly Leu Thr Gly Asn Ile Phe Glu Val
115 120 125
Phe Leu Lys Pro Tyr Phe Leu Glu Ala Tyr Arg Pro Ile His Lys Gly
130 135 140
Asp Leu Phe Leu Val Arg Gly Gly Met Arg Ala Val Glu Phe Lys Val
145 150 155 160
Val Glu Thr Asp Pro Ala Pro Tyr Cys Ile Val Ser Gln Asp Thr Val
165 170 175
Ile Tyr Cys Glu Gly Glu Pro Val Lys Arg Glu Glu Glu Glu Glu Gln
180 185 190
Leu Asn Glu Val Gly Tyr Asp Asp Ile Gly Gly Cys Arg Lys Gln Leu
195 200 205
Ala Gln Ile Lys Glu Met Val Glu Leu Pro Leu Arg His Pro Ser Leu
210 215 220
Phe Lys Ala Ile Gly Val Lys Pro Pro Arg Gly Ile Leu Leu Tyr Gly
225 230 235 240
Pro Pro Gly Thr Gly Lys Thr Leu Ile Ala Arg Ala Val Ala Asn Glu
245 250 255
Thr Gly Ala Phe Phe Phe Leu Ile Asn Gly Pro Glu Ile Met Ser Lys
260 265 270
Leu Ala Gly Glu Ser Glu Ser Asn Leu Arg Lys Ala Phe Glu Glu Ala
275 280 285
Glu Lys Asn Ser Pro Ala Ile Ile Phe Ile Asp Glu Ile Asp Ala Ile
290 295 300
Ala Pro Lys Arg Glu Lys Thr His Gly Glu Val Glu Arg Arg Ile Val
305 310 315 320
Ser Gln Leu Leu Thr Leu Met Asp Gly Leu Lys Gln Arg Ser His Val
325 330 335
Ile Val Met Ala Ala Thr Asn Arg Pro Asn Ser Ile Asp Pro Ala Leu
340 345 350
Arg Arg Phe Gly Arg Phe Asp Arg Glu Val Asp Ile Ser Ile Pro Asp
355 360 365
Thr Thr Gly Arg Leu Glu Val Leu Arg Ile His Thr Lys Asn Met Lys
370 375 380
Leu Ser Asp Asp Val Asp Leu Glu Gln Ile Ala Ala Glu Thr His Gly
385 390 395 400
His Val Gly Ala Asp Leu Ala Ala Leu Cys Ser Glu Ala Ala Leu Gln
405 410 415
Gln Ile Arg Glu Lys Met Asp Leu Ile Asp Leu Asp Asp Asp Gln Ile
420 425 430
Asp Ala Glu Val Leu Asn Ser Leu Ala Val Thr Met Glu Asn Phe Arg
435 440 445
Phe Ala Met Gly Lys Ser Thr Pro Ser Ala Leu Arg Glu Thr Val Val
450 455 460
Glu Val Pro Asn Ile Thr Trp His Asp Ile Gly Gly Leu Glu Asn Val
465 470 475 480
Lys Arg Glu Leu Gln Glu Leu Val Gln Tyr Pro Val Glu His Pro Asp
485 490 495
Lys Phe Leu Lys Phe Gly Met Thr Pro Ser Lys Gly Val Leu Phe Tyr
500 505 510
Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala Lys Ala Ile Ala Asn
515 520 525
Glu Cys Gln Ala Asn Phe Ile Ser Ile Lys Gly Pro Glu Leu Leu Thr
530 535 540
Met Trp Phe Gly Glu Ser Glu Ala Asn Val Arg Asp Val Phe Asp Lys
545 550 555 560
Ala Arg Ala Ala Ala Pro Cys Val Leu Phe Phe Asp Glu Leu Asp Ser
565 570 575
Ile Ala Lys Ala Arg Gly Gly Ser Ala Gly Asp Ala Gly Gly Ala Ala
580 585 590
Asp Arg Val Ile Asn Gln Val Leu Thr Glu Met Asp Gly Met Gly Ala
595 600 605
Lys Lys Asn Val Phe Ile Ile Gly Ala Thr Asn Arg Pro Asp Ile Ile
610 615 620
Asp Pro Ala Ile Leu Arg Pro Gly Arg Leu Asp Gln Leu Ile Tyr Ile
625 630 635 640
Pro Leu Pro Asp Glu Lys Ser Arg Val Gln Ile Leu Lys Ala Cys Leu
645 650 655
Arg Lys Ser Pro Val Ser Lys Arg Val Asp Leu Glu Tyr Leu Ala Lys
660 665 670
Val Ser His Gly Phe Ser Gly Ala Asp Leu Thr Glu Ile Cys Gln Arg
675 680 685
Ala Cys Lys Leu Ala Ile Arg Gln Ala Ile Glu Ala Asp Ile Lys Arg
690 695 700
Glu Arg Glu Arg Ala Ala Gly Asp Asn Met Asp Met Glu Glu Glu Asp
705 710 715 720
Pro Val Pro Glu Ile Thr Arg Asp His Phe Glu Glu Ala Met Lys Tyr
725 730 735
Ala Arg Arg Ser Val Ser Asp Asn Asp Ile Arg Lys Tyr Glu Met Phe
740 745 750
Ser Gln Thr Leu Gln Gln Ser Arg Gly Phe Gly Ser Asn Phe Arg Phe
755 760 765
Pro Asp Gln Gln Gly Gln Gly Gly Ser Ser His Gly Gly Asn Phe Gly
770 775 780
Ala Asp Gly Glu Asp Asp Asp Leu Tyr Ser
785 790
Claims (5)
1. the gene order of red claw crayfish valosin-containing protein is：
2. red claw crayfish valosin-containing protein amino acid sequence is：
3. the preparation method of red claw crayfish valosin-containing protein, it is characterised in that comprise the following steps：
1) Cq-VCP genes are connected to carrier pMD18-T, and analyze its open reading frame, obtain Cq-VCP gene orders；
2) the Cq-VCP gene orders obtained by step 1), carry out phylogenetic analysis and analyze its protein structure domain.
4. the preparation method of red claw crayfish valosin-containing protein as claimed in claim 3, it is characterised in that the Cq-VCP is three
A member in adenosine phosphate enzyme superfamily, it is with typical domain.
5. red claw crayfish valosin-containing protein is applied in Anti-infection to WSSV medicine is prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032401.3A CN108018289B (en) | 2018-01-12 | 2018-01-12 | Gene Cq-VCP for inhibiting WSSV early infection and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032401.3A CN108018289B (en) | 2018-01-12 | 2018-01-12 | Gene Cq-VCP for inhibiting WSSV early infection and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108018289A true CN108018289A (en) | 2018-05-11 |
| CN108018289B CN108018289B (en) | 2020-07-14 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109055411A (en) * | 2018-09-18 | 2018-12-21 | 厦门大学 | Inhibit half Guang Aspartase gene C q-caspase and its albumen antiviral activity application of WSSV proliferation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106177961A (en) * | 2016-08-18 | 2016-12-07 | 广州威溶特医药科技有限公司 | The application in preparing antitumor drug of VCP inhibitor and oncolytic virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106177961A (en) * | 2016-08-18 | 2016-12-07 | 广州威溶特医药科技有限公司 | The application in preparing antitumor drug of VCP inhibitor and oncolytic virus |
Non-Patent Citations (3)
| Title |
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| DEBASIS PANDA ET AL.: "Genome-wide RNAi screen identifies SEC61A and VCP as conserved regulators of Sindbis virus entry", 《CELL REP》 * |
| RONG-YUAN CHEN ET AL.: "White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner", 《SCIENTIFIC REPORTS》 * |
| WITCHULADA TALAKHUN ET AL.: "Characterization，expression and localization of valosin-containing protein in ovaries of the giant tiger shrimp Penaeus monodon", 《GENE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055411A (en) * | 2018-09-18 | 2018-12-21 | 厦门大学 | Inhibit half Guang Aspartase gene C q-caspase and its albumen antiviral activity application of WSSV proliferation |
| CN109055411B (en) * | 2018-09-18 | 2022-02-11 | 厦门大学 | Caspase gene Cq-caspase for inhibiting WSSV proliferation and application of protein antiviral activity thereof |
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| CN108018289B (en) | 2020-07-14 |
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