CN110195093A - A kind of medicament sifting motion system for recombinating zika virus and its application based on expressing green fluorescent protein - Google Patents
A kind of medicament sifting motion system for recombinating zika virus and its application based on expressing green fluorescent protein Download PDFInfo
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Abstract
The medicament sifting motion system for the recombination zika virus that the invention discloses a kind of based on expressing green fluorescent protein and its application.A kind of medicament sifting motion system of the invention is made of the recombination zika virus (report type zika virus) of expressing green fluorescent protein (GFP) and the BHK-21 cells of expression DC-SIGNR.Report type zika virus expressing green fluorescent protein of the invention, fluorescence number is strong, and the uniform coloring in endochylema, is easy to observe, and is easy to high flux screening analysis.The BHK-21 cells for stablizing expression DC-SIGNR improve the sensibility infected report type zika virus, improve viral replication efficiency, reduce virus replication pressure, also improve the stability of recombinant virus.Medicament sifting motion system through the invention can carry out directly observation and analysis to the virus replication situation before and after drug effect under living cells state, it does not need cell is fixed and immunostaining, keep entire operation process more simple and efficient, and reduce live virus operating procedure, improve the safety of entire operation process.
Description
Technical field
It is the present invention relates to a kind of screening system of antiviral drugs and its application, in particular to a kind of glimmering based on expression green
The medicament sifting motion system of the recombination zika virus of photoprotein and its application.The invention belongs to field of biotechnology.
Background technique
Zika virus (Zika virus, ZIKV) is a kind of Amphixenosis's cause of disease mainly propagated through mosquito matchmaker, and is stepped on
It removes from office fever virus (Dengue virus, DENV), west nile virus (West Nile virus, WNV), Japanese B encephalitis virus
(Japanese encephalitis virus, JEV) and yellow fever virus (Yellow Fever virus, YFV) etc. belong to jaundice
Malicious section's Flavivirus member.Nineteen forty-seven, the researchers such as Dick are isolated in the rhesus monkeys in stockaded village, Uganda Carson woods
Out.Stockaded village's card can be caused hot after ZIKV infection host, most of the infecteds are there is no manifest symptom or symptom are more slight, packet
Include the symptoms such as conjunctivitis, fash, fever, joint and myalgia, the course of disease about 2-7 days.If but ZIKV sense during gestation occurs
Dye may then make newborn with microcephaly and other congenital abnormalities, while may also result in pregnant person and premature labor and stream occurs
The serious symptoms such as production.There are about 86 countries, there are ZIKV in the world for studies have shown that, this shows ZIKV in the world
In spreading trend.A few days ago without the vaccine and antiviral drugs of approval application.For more effectively prevention and control disease, need to take more
Kind advanced technology means carry out vaccine research and drug screening.Report type virus is constructed with reverse genetic operating system to accelerate
The vaccine research of the virosis, report type virus also provide important technological platform for drug screening, are the effective of virological investigation
Advanced means.But the unstability of the recombinant virus due to the unstability and foreign gene-carrying of genome, in jaundice
In poison, constructing stable report type virus is always a problem and challenge.
ZIKV virion is spherical in shape, diameter about 40~70nm, have outside virion one layer from host derivation
Lipid cyst membrane package, genome are the single-stranded positive RNA of non-segmented negative, and length about 11kb, encoding gene sequence is 5 '-C-
prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'.RNA is translated as a polyprotein, then passes through protease
It is cracked into structural proteins capsid protein (capsid, C), precursor memebrane protein (precursor membrane, prM), envelope protein
Seven kinds of non-structural proteins of (envelope, E) and NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5.Wherein C protein combines
ZIKV geneome RNA forms nucleocapsid core, and prM albumen and E protein then form virus surface proteins, and in host cell,
Non-structural protein then participates in the processes such as virus replication and polyprotein shearing.Currently, according to molecular biology and biology letter
Breath, which is learned, can be classified as two kinds of hypotypes of Asian type and African type.Construct stable viral genomic clone and have difficulties be due to
Exist in virus genomic E protein gene and NS1 protein gene and has virose coded sequence to host strain.And research is aobvious
Show, is deposited by the report type zika virus that reverse genetics manipulation technology constructs expressing luciferase gene or fluorescence protein gene
The case where replication capacity declines, and exogenous gene expression is unstable, and foreign gene is lost rapidly after passage.
The present invention constructs the recombination zika virus of expressing green fluorescent protein (GFP) by the method for in-vivo recombination
ZIKV-GFP.And construct the cell line of one plant of expression DC-SIGNR.The cell line promotes report type zika virus ZIKV-
The duplication of GFP reduces the inheritance stability pressure of recombinant virus, improves the stability of recombination report type virus.Recombination disease
Poison constitutes a convenient zika virus medicament sifting motion system with stable expression cell line, can be used for the medicine of anti-zika virus
Object screening can be also used for the neutralizing antibody detection of zika virus, can be used for the evaluation of zika virus vaccine effect.
Summary of the invention
The purpose of the present invention is to provide a kind of systems for anti-zika virus drug screening, to overcome report type stockaded village card
The defects of replication capacity existing for virus is low, unstable.
In order to achieve the above object, present invention employs following technological means:
One kind of the invention is based on the recombination zika virus (Zika virus, ZIKV) of expressing green fluorescent protein (GFP)
Medicament sifting motion system, the medicament sifting motion system by expressing green fluorescent protein recombination zika virus and expression DC-
The BHK-21 cells of SIGNR form.
Wherein, it is preferred that the recombination zika virus of the expressing green fluorescent protein is to be prepared by the following method
It arrives:
1) it according to zika virus gene order, is synthesized by segmented genes synthetic technology other in addition to structural protein gene
The GFP reporter sequences gene of the structural protein gene position of genome sequence and insertion and deletion, passes through polyclonal digestion
Site is connected into mammalian expression vector, obtains replicon recombinant plasmid, and the open reading frame of the replicon recombinant plasmid is
" CMV promoter-restriction enzyme site -5 ' UTR- coding 20 amino acid of C protein N-terminal nucleotide sequence-GFP-FMDV2A- encodes E
Nucleotide sequence-NS1~5 (non-structural protein 1~5 gene orders) -3 ' UTR-HDVr (fourth liver of 22, PROTEIN C end amino acid
Virus ribozymal gene order)-restriction enzyme site "；
2) according to zika virus gene order, by gene synthesis technology be respectively synthesized structural protein gene sequence and
GFP sequence, the order of connection of each element are the nucleotide sequence-GFP- of 5 ' UTR-coding ZIKV C protein C-terminal, 38 amino acid
The segment is cloned into lactation by polyclone enzyme enzyme site by 1-134 nucleotide sequences of FMDV2A-C-prM-E-NS1 gene
On animal expression vector, the plasmid containing ZIKV structural gene and green fluorescent protein is obtained；
3) rescue of report type ZIKV
Using the obtained plasmid of step 2) as template, PCR amplification is carried out, amplification, which obtains, contains CMV promoter and 5 '
UTR-coding ZIKV C protein 38 amino acid of C-terminal nucleotide sequence-GFP-FMDV2A-C-prM-E-NS1 gene 1-134
The PCR product of nucleotide sequence, PCR product are used to report the rescue of type virus；
The rescue of report type ZIKV: HEK-293T cell in 24 orifice plates use the culture of 10%v/v FBS DMEM culture medium extremely
Density is 70%-90%, using TransIT-293Transfection Reagent transfection reagent by obtained PCR product and
The replicon recombinant plasmid of linearisation is transfected respectively, after transfecting 72h, is harvested cell and supernatant and is saved in -70 DEG C, obtain table
Up to the recombination zika virus of green fluorescent protein.
Wherein, it is preferred that the zika virus gene order is the gene order of the popular ZIKV strain of Brazil in 2015,
Genbank accession number are as follows: KX280026.
Wherein, it is preferred that be that mammal expression is connected by polyclone enzyme enzyme site Sac I and Not I in step 1)
Carrier pCI-neo, obtains replicon recombinant plasmid, which is followed successively by CMV promoter-Sac
Nucleotide sequence-the GFP-FMDV2A- that I-5 ' UTR- encodes 20 amino acid of C protein N-terminal encodes 22 amino acid of E protein C-terminal
Nucleotide sequence-NS1~5 (non-structural protein 1~5 gene orders) -3 ' UTR-HDVr (fourth hepatovirus ribozyme gene sequence) -
Not I。
Wherein, it is preferred that be by polyclone enzyme enzyme site XhoI and NotI in step 2) by 5 ' UTR-coding ZIKV C
1-134 nucleotide sequence clones of nucleotide sequence-GFP-FMDV2A-C-prM-E-NS1 gene of 38, PROTEIN C end amino acid
Onto pCI-neo carrier.
Wherein, it is preferred that be using the obtained plasmid of step 2) as template, with primer P1:5 '-in step 3)
GGCCTTTTGCTCACATGGCTCGACAG-3 ' and P2:5 ' GACTGCTGCTGCCAATCTACGGGGG-3 ' carries out PCR amplification,
PCR product is used to report the rescue of type virus.
Wherein, it is preferred that the BHK-21 cells of the expression DC-SIGNR construct by the following method to be obtained:
1) expression plasmid containing DC-SIGNR is constructed；
2) the DC-SIGNR expression plasmid prepared is linearized, after glue recycles, BHK-21 cell is turned
Dye；
3) screening obtains the BHK-21 cells for stablizing expression DC-SIGNR.
Wherein, it is preferred that the coding nucleotide sequence of DC-SIGNR is as shown in SEQ ID NO.1, by the DC- after synthesis
SIGNR sequence is cloned into pCAGneo carrier.
Further, the invention also provides the medicament sifting motion systems to screen answering in anti-zika virus drug
With.
Further, anti-zika virus drug is screened using the medicament sifting motion system the invention also provides a kind of
Method, comprising the following steps:
1) drug-treated and virus infection: the BHK-21 cells for expressing DC-SIGNR are cultivated with 2%v/vFBS100ul
Base is inoculated in 96 orifice plate of black, when cell density is up to 90%, is diluted with the DMEM culture medium of 50ul FBS containing 2%v/v
The recombination zika virus infection of expressing green fluorescent protein, in 37 DEG C, 5%CO are added after drug effect 1h to be measured, 1h2Incubator
Middle culture 48h；
2) high intension system statistical analysis: cell is after drug-treated and virus infection 48h, with Hoechst staining cell
Cell plates are placed in high intension cell screening system (PerkinElmer) and observed by core, using green fluorescent protein fluorescence channel with
Nuclear targeting fluorescence channel scanning analysis counts every hole infection positive cell number and total cell number, calculates viral infection rate, meter
Drug to be measured is calculated to the inhibiting rate of virus replication.
Compared to the prior art, the beneficial effects of the present invention are:
The report type flavivirus constructed with reverse genetics system expresses fluorescin such as expressing luciferase recombinant virus
Recombinant virus it is unstable, and replication capacity weakens significantly, and under this duplication pressure, foreign gene is easily lost, and restores
Sport wild-type virus.Present system constructs the cell line for stablizing expression DC-SIGNR molecule, which improves pair
The sensibility of report type zika virus infection, improves viral replication efficiency, reduces virus replication pressure, also improve recombination
The stability of virus.Reporter virus expressing green fluorescent protein of the invention, fluorescence number is strong, and the uniform coloring in endochylema, easily
In observation, it is easy to high flux screening analysis.Report type virus system of the invention can be in living cells shape to virus replication situation
Intuitively observation and analysis under state, does not need cell is fixed and immunostaining, keeps entire operation process more simple and efficient, and
Reduce live virus operating procedure, improves the safety of entire operation process.
Detailed description of the invention
Fig. 1 is the identification of pCAG-DC-SIGNR plasmid；
M1:DL 10000DNA standard；1:Sac I and Xho I digestion identification；2:pCAG-neo plasmid；
Fig. 2 is ZIKV replicon pZIKVrepdCME and pC38GFP-CME-NS1134The building schematic diagram of plasmid；
Fig. 3 is ZIKV replicon pZIKVrepdCME (A), pC38GFP-CME-NS1134(B) knot is identified in the digestion of plasmid
Fruit；
Wherein in Fig. 3 A, M1: DNA maker, 1: non-digested plasmid compares；2:Xhol I and Not I double digestion identification knot
Fruit；
Wherein in Fig. 3 B, M2: DNA maker, 1:Sac I and Not I double digestion qualification result；2: non-digested plasmid pair
According to；
The Western blot that Fig. 4 is recombinant virus ZIKV-GFP is identified；
M: protein molecular weight standard；1: virus infected cell；2: viral non-infected cells；
Fig. 5 is the observation of recombinant virus ZIKV-GFP expressing green fluorescent protein；
Fig. 6 is the plaque formation that recombinant virus ZIKV-GFP infects BHK-21, Vero and BHK-DR cell；
Fig. 7 is recombinant virus ZIKV-GFP growth kinetics curve determination；
Fig. 8 is the anti-ZIKV drug study of Ribavirin；
Wherein Fig. 8 A is using green fluorescent protein fluorescence channel and nuclear targeting fluorescence channel scanning analysis result；Figure
8B is viral infection rate statistical result；Fig. 8 C is inhibiting rate of the drug to virus replication；
Fig. 9 is the anti-ZIKV drug study of 6-Azauridine；
Wherein Fig. 9 A is using green fluorescent protein fluorescence channel and nuclear targeting fluorescence channel scanning analysis result；Figure
9B is viral infection rate statistical result；Fig. 9 C is inhibiting rate of the drug to virus replication；
Figure 10 is the anti-ZIKV drug study of Chloroquine.
Wherein Figure 10 A is using green fluorescent protein fluorescence channel and nuclear targeting fluorescence channel scanning analysis result；
Figure 10 B is viral infection rate statistical result；Figure 10 C is inhibiting rate of the drug to virus replication.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1 stablizes the building of expression DC-SIGNR cell line
1.1 experimental material
1.1.1 main material and reagent
BHK-21 cell；DMEM, fetal calf serum, Opti-MEM are purchased from Gibco company；Pancreatin and the purchase of mycillin mixed liquor
From Gibco company；Poly-D-lysine is purchased from Sigma company；TritoX-100 is purchased from Sigma company；Infrared fluorescent labels resist
Rabbit igg antibody is purchased from KPL company；The goat anti-mouse IgG of FITC label is purchased from middle mountain gold bridge company；Qiagen plasmid extracts examination
Agent box is purchased from QIAGEN company；TransIT-293Transfection Reagent is purchased from Mirus company.
1.1.2 laboratory apparatus
PCR instrument (Takara)；Nucleic acid electrophoresis apparatus (6 1 instrument of Beijing)；Nucleic acid gel imaging system (Bio-Rad)；Pure water
Instrument (Millipore)；Optical microscopy (Olympus)；Inverted fluorescence microscope (Life)；Electronic balance (Beijing Sai Duoli
This)；CO2 constant incubator (Heal Force)；Standard pipettor (Eppendorf)；Horizontal centrifuge (Eppendorf)；
1.2 experimental method
1.2.1 the building of DC-SIGNR plasmid is expressed
According to the DC-SIGNR gene coded sequence (Genbank accession number are as follows: AY042234) that GenBank is announced, to it
Carry out artificial synthesized, the sequence after synthesis is as shown in SEQ ID NO.1.Sequence after synthesis is cloned into pCAGneo carrier (should
Carrier has been documented in " stablizing the foundation of expression japanese encephalitis virus NS1 albuminous cell system, Chen Zhenshi, master thesis " text
In, provided by Harbin veterinary institute Vet Biotechnology National Key Laboratory) in, building pCAG-DC-SIGNR expression
Plasmid.
1.2.2pCAG-DC-SIGNR the extraction of plasmid
Strain containing pCAG-DC-SIGNR plasmid is subjected to scribing line recovery, picking monoclonal colonies in 4ml LB into
Then row shaken cultivation therefrom takes 1ml bacterium solution to be inoculated into shaken cultivation 4h in 100ml triangular flask according to 1:100 ratio column.Collect bacterium
After liquid, plasmid extraction is carried out with qiagen plasmid extracts kit, extraction step is as follows: being harvested from LB culture medium and be incubated overnight
The bacterium solution of 12-16h is centrifuged 5min under the conditions of 10000rpm, 4 DEG C in 50ml centrifuge tube；Supernatant of bacteria solution after being centrifuged is discarded, is added
Enter 10ml Buffer P1 and bacterium is resuspended；After bacterium is resuspended completely, 10ml Buffer P2 is added, acutely shakes centrifuge tube 4-
Mix it thoroughly 6 times, room temperature acts on 5min, if having used LyseBlue reagent in P1, solution should become uniform at this time
Blue；In incubation period, opens the sealing cover of needle mouth and filter is put on suitable shelf；10ml ice bath is added
For Buffer P3 into this lysate, acutely shaking centrifuge tube 4-6 times makes its mixing, if joined LyseBlue examination in P1
Agent then mixes reagent until not having color；Lysate is centrifuged 10min under the conditions of 6000g, 4 DEG C；It is centrifuged in lysate
It is period, QIAGEN-tip column is vertical on the support frame, 10ml Buffer QBT is added, solution in pipe is allowed to flow through by gravity；To
After the completion of QBT solution equilibria pillar, the limpid supernatant addition of lysate is balanced in complete QIAGEN-tip pillar and has allowed it
It is slowly dripped through resin film by gravity；After the completion of previous step, QIAGEN-tip column 2 is washed with 10ml Buffer QC
It is secondary；Eluted dna (is incubated) in 65 DEG C of water in 10ml centrifuge tube after the completion of previous step, then with 5ml Buffer QF；It is added
2.5ml isopropanol mixes well in eluent, and DNA is made to precipitate, with 12000rpm, 4 DEG C of centrifugation 30min；Wait be centrifuged
Cheng Hou gently discards supernatant, DNA precipitating is resuspended with 70% ethyl alcohol of 2ml, with 12000rpm, 4 DEG C of centrifugation 10min；Wait be centrifuged
Supernatant carefully is poured out at rear, plasmid is not encountered and keep plasmid complete, the plasmid of centrifuge tube bottom be air-dried in super-clean bench, about
15min；DNA is re-dissolved with the ddH2O of the endotoxin-free of suitable volumes；The concentration of detection DNA plasmid is placed in -20 DEG C of guarantors
It deposits.
1.2.2 the transfection of plasmid
The pCAG-DC-SIGNR expression plasmid prepared is linearized into (table 1), after glue recycles, is utilized
TransIT-293Transfection Reagent is transfected to specifications.
The linearisation system of 1 carrier for expression of eukaryon pCAG-DC-SIGNR of table
After glue recycles, well-grown BHK-21 cell dissociation is got off, 24 orifice plates are inoculated in, it is long extremely to cell
It is transfected according to the following steps when 70%-90%: opti-MEM and TransIT-293Transfection Reagent is taken in 4 DEG C
Out, it is placed in room temperature about 20min, while taking out and having linearized complete pCAG-DC-SIGNR plasmid；Take 100 μ l opti-MEM in
In 1.5ml EP pipe, 2 μ g pCAG-DC-SIGN linearization plasmids are added, is gently vortexed and mixes；Take 3 μ l TransIT-
293Transfection Reagent is added in the mixed liquor that previous step has mixed, and is gently vortexed and is mixed；15min to be incubated for is left
It behind the right side, is added in 24 orifice plates with every 50 μ l mixed liquor of hole, 24-72h observes result.
1.2.3 screening and the clone purification of cell are transfected
After 48h to be transfected, screening and the clone purification of cell are carried out in accordance with the following steps: after transfection cell is digested
And it counts；It carries out calculating required total number of cells amount according to the quantity of every 1 cell in hole；96 orifice plates are taken, every hole is added 100 μ l and contains
The culture medium of G418 and 10%v/v FBS simultaneously makes every hole cell quantity 0.5-1；Culture 5 days or so is under inverted microscope
Cell quantity is observed, and records the cell colony grown up to containing individual cells；The cell is subjected to IFA identification, is completed with this
Screening and clone purification.
1.3 experimental result
1.3.1DC-SIGNR the building of expression plasmid
By pCAG-DC-SIGNR plasmid after the identification of Sac I and Xho I double digestion, occur at 1218bp as the result is shown
Obvious purpose band (Fig. 1), successfully constructs pCAG-DC-SIGNR expression plasmid.
1.3.2 the IFA identification of cell is transfected
Cell is transfected to be screened and carry out IFA detection after clone purification.The cell clone screened is in endochylema and cell membrane
Express DC-SIGNR on surface, and control cell is then without specific fluorescence signal.The result shows that: by G418 screening and IFA mirror
Fixed, the present invention successfully filters out the cell line for stablizing expression DC-SIGNR, which is named as BHK-DR cell.
Embodiment 2: the building of the report type zika virus ZIKV-GFP of expressing green fluorescent protein
2.1 experimental material
2.1.1 main material, reagent
HEK-293T, Vero, BHK-21 cell are saved by this laboratory；DMSO is purchased from Sigma company；DMEM, tire ox blood
Clearly, Opti-MEM is purchased from Gibco company；Pancreatin (TRYPSIN 0.25%EDTA) and blueness-streptomysin mixed liquor are public purchased from Gibco
Department；Poly-D-lysine is purchased from Sigma company；Hoechst is purchased from Pierce company；Triton X-100 is purchased from Sigma company；It is red
The anti-rabbit IgG antibody of outer fluorescent marker is purchased from KPL company；The goat anti-mouse IgG of FITC label is purchased from middle mountain gold bridge company；
Qiagen plasmid extracts kit is purchased from QIAGEN company；TransIT-293Transfection Reagent is public purchased from Mirus
Department；Chloroquine (Chloroquin) and 6- azauridine (6-Azauridine) are purchased from Sigma company；ZIKV E protein antibody is purchased from
GeneTex company.
2.1.2 laboratory apparatus
High intension cell screening system (PerkinElmer)；PCR instrument (Takara)；Nucleic acid electrophoresis apparatus (6 1 instrument of Beijing
Device)；Nucleic acid gel imaging system (Bio-Rad)；Pure water meter (Thermo Fisher)；Optical microscopy (Olympus)；It is inverted
Fluorescence microscope (Life)；Electronic balance (Beijing Sai Duolisi)；CO2 constant incubator (Heal Force)；Standard pipettor
(Eppendorf)；Horizontal centrifuge (Eppendorf)；Protein electrophoresis instrument (Bio-Rad)；Half dried albumen transferring film instrument (Bio-
Rad)；Protein gel imaging system (Odyssey).
2.2 experimental method
2.2.1ZIKV replicon and pC38GFP-CME-NS1134The building of plasmid
According to Brazilian prevalence ZIKV pnca gene sequence (Genbank accession number are as follows: KX280026) in 2015, by being segmented base
Because of the structural protein gene position of other genome sequences and insertion and deletion of the synthetic technology synthesis in addition to structural protein gene
GFP reporter sequences gene, mammalian expression vector pCI- is connected by polyclone enzyme enzyme site Sac I and Not I
Neo obtains replicon recombinant plasmid pZIKVrepdCME.The replicon plasmid open reading frame is followed successively by " CMV promoter-Sac
Nucleotide sequence-the GFP-FMDV2A- that I-5 ' UTR- encodes 20 amino acid of C protein N-terminal encodes 22 amino acid of E protein C-terminal
Nucleotide sequence-NS1~5 (non-structural protein 1~5 gene orders) -3 ' UTR-HDVr (fourth hepatovirus ribozyme gene sequence) -
Not I ", replicon plasmid construction process are shown in Fig. 2.Meanwhile according to ZIKV gene order, it is respectively synthesized by gene synthesis technology
" 5 ' UTR-C38 (nucleotide sequence of coding 38 amino acid of ZIKV C protein C-terminal)-GFP (green fluorescence protein gene sequence
Column)-FMDV2A-C-prM-E (ZIKV structural protein gene sequence)-NS1134(1-134 nucleotide sequences of NS1 gene) " pass through
The segment is cloned on pCI-neo carrier to construct pC38GFP-CME-NS1 by polyclone enzyme enzyme site XhoI and NotI134Matter
Grain (Fig. 2).
2.2.2 the rescue of report type ZIKV
With plasmid pC38GFP-CME-NS1134For template, with primer P1:5 '-GGCCTTTTGCTCACATGGCTCGACAG-
3 ' carry out PCR amplification with P2:5 ' GACTGCTGCTGCCAATCTACGGGGG-3 ', and PCR product is used to report the rescue of type virus.
The rescue of report type ZIKV: HEK-293T cell in 24 orifice plates use the culture of 10%v/vFBS DMEM culture medium extremely
Density is 70%-90%, using TransIT-293Transfection Reagent transfection reagent by pC38GFP-CME-
NS1134Plasmid PCR product and Xhol I linearisation pZIKVrepdCME replicon plasmid respectively with the ratio of every hole 500ng into
Row transfection.After transfecting 72h, harvests cell and supernatant and saved in -70 DEG C.It is ZIKV-GFP by the viral nomenclature of rescue.
2.2.3 the identification of recombinant virus
2.2.3.1 Electronic Speculum observation identification
Normal passage BHK-DR cell is in T25 cell bottle, and to cell density to 70%-90%, ZIKV-GFP is with MOI=1
BHK-DR cell is infected, while the control of BHK-DR cell blank is set, 72h is cultivated in 37 DEG C, 5%CO2 incubator, through fixation
After liquid processing, it is made into section sample, uses electron microscope observation virion.
2.2.3.2Western blot and IFA is detected
The ZIKV-GFP that the present invention constructs is inoculated in BHK-DR cell with MOI=1, while BHK-DR cell blank is set
Control carries out Western blot detection after 72h.It is thin that the ZIKV-GFP that constructs of the present invention with MOI=1 is inoculated in BHK-DR
Born of the same parents, while the control of BHK-DR cell blank is set, IFA detection is carried out after 48h.
2.2.3.3 recombinant virus infection BHK-DR cell forms plaque
BHK-21, Vero and BHK-DR cell with 2%FBS 500ul culture medium respectively after 24 orifice plates normally pass on, to
When cell density is to 90%, the viral ZIKV-GFP that the present invention is constructed is with 10-1、10-2、10-3、10-4、10-5Gradient dilution is simultaneously
It is added with the every hole 100ul into 24 orifice plates.We add 3% methylcellulose of 600ul (15g first after infection cell in 6-12h
DMEM liquid after the sterilizing of base cellulose with 500mL containing 1% cow's serum sufficiently dissolves) covering cell.Meanwhile being arranged in experiment and not feeling
Dye BHK-DR cell hole is negative control.Covering is discarded after 7d adds the every hole 1mL of 0.1% crystal violet after PBS is rinsed one time,
Dyeing 20-30 minutes, is gently rinsed with flowing water, and inversion observes and records plaque formation situation after drying.
2.2.3.4ZIKV-GFP growth kinetics curve determination
The ZIKV-GFP that the present invention constructs is inoculated in BHK-21 cell, Vero cell, BHK-DR with MOI=0.1 respectively
Cell, after infection for 24 hours, 48h, 72h, 96h, 120h, 144h continuous sampling in -20 DEG C save.Collect the ZIKV of each period with
The sample of ZIKV-GFP carries out plaque counting experiment, and the ZIKV-GFP sample that each period is collected then is being inverted fluorescence microscopy
It is counted under mirror, growth curve is drawn respectively with this.
2.3 result
2.3.1ZIKV replicon and pC38GFP-CME-NS1134The building of plasmid
According to ZIKV gene order, other genome sequences and insertion and deletion of the building in addition to structural protein gene
The GFP reporter sequences gene of structural protein gene position, after the identification of Xhol I and Not I double digestion, successfully building is multiple
System recombinant plasmid pZIKVrepdCME (Fig. 3 A).According to ZIKV gene order, pC38GFP-CME-NS1 is constructed134Plasmid, warp
After the identification of Sac I and Not I double digestion, pC38GFP-CME-NS1 is successfully constructed134Plasmid (Fig. 3 B).
2.3.2 the Electronic Speculum identification of ZIKV-GFP is recombinated
ZIKV-GFP is made into section sample after fixer is handled with MOI=1 infection BHK-DR cell, uses electronic display
Micro mirror observes virion, as the result is shown: the BHK-DR cell of ZIKV-GFP infection can find recombinant virus in endoplasmic reticulum
Particle.
2.3.3ZIKV Western blot and the IFA detection of structural proteins
The ZIKV-GFP that this laboratory constructs is inoculated in BHK-DR cell, and the rabbit prepared with this laboratory with MOI=1
Polyclonal antibody carries out IFA and Western blot respectively and detects, and the rabbit anteserum obtained after immune using 3 times is primary antibody, infrared
The goat anti-rabbit igg of fluorescent marker be secondary antibody carry out western blot experiment, with ZIKV E protein specific polyclonal antibody into
Row detection, as a result occurs specific band (Fig. 4) at relative molecular mass 55kDa, is consistent with expected results.Meanwhile it is right
Recombinant virus infection cell carries out green fluorescent protein observation, and as a result infection cell generates bright green fluorescence signal, and negative right
(Fig. 5) is generated according to then unstressed configuration signal, shows expression of recombinant virus green fluorescent protein, virus infected cell is played well
Report is acted on label.
2.3.4 the formation plaque of recombinant virus ZIKV-GFP infection cell
The ZIKV-GFP virus that the present invention is constructed is with 10-1、10-2、10-3、10-4、10-5Gradient dilution is simultaneously every with 100ul
Hole is added into BHK-21, Vero and BHK-DR cell for being grown to 90% density.We add in 6-12h after infection cell
3% methylcellulose of 600ul covers cell.Covering is discarded after 7d adds the every hole of 0.1% crystal violet after PBS is rinsed one time
After dyeing 20-30min, discovery ZIKV-GFP can form Virus plaque in BHK-DR cell and Vero cell, and in BHK-21
Plaque (Fig. 6) is had no in cell.
2.3.5 recombinant virus ZIKV and ZIKV-GFP growth kinetics curve determination
It is thin that the ZIKV that constructs of the present invention is inoculated in MOI=0.1 to BHK-21 cell, Vero cell and BHK-DR respectively
Born of the same parents, after infection for 24 hours, 48h, 72h, 96h, 120h, 144h continuous sampling carry out plaque counting experiment.Meanwhile ZIKV-GFP is with MOI
=0.1 is inoculated in Vero cell and BHK-DR cell, and the ZIKV-GFP sample that above each period is collected is then glimmering in inversion
Counted under light microscope, growth curve drawn respectively with this, as the result is shown: ZIKV and ZIKV-GFP between -72h for 24 hours,
Virus titer is totally in rising trend, and reaches maximum to 72h virus titer, totally on a declining curve to 96h-144h, so really
It is fixed to receive malicious Best Times as 72h after infection or so (Fig. 7).
3 report type virus system of embodiment is screened for antiviral drugs
3.1 antiviral drugs Inhibition tests
Drug: drug Ribavirin (Rib) and 6-Azauridine (6-Az) are dissolved with DMSO, chloroquine
(Chloroquine, CQ) is dissolved with PBS, and DMSO is final concentration of 1% in culture solution, and not plus pharmaceutically-active cell is with 1%
DMSO is used as negative control.Drug is diluted to various concentration with DMSO and the DMEM culture medium containing 2% fetal calf serum (FBS)
For use.
Drug-treated and virus infection: by BHK-DR cell (3 × 104A/hole) it is connect with 2%v/vFBS 100ul culture medium
Kind in 96 orifice plate of black, when cell density is up to 90%, the drug that was diluted with the DMEM culture medium of 50ul FBS containing 2%v/v
1h is acted on, 50ul ZIKV-GFP virus (10 is added after 1h6TCID50/ ml) infection, in 37 DEG C, 5%CO2It is cultivated in incubator
48h。
High intension system statistical analysis: cell is contaminated after drug-treated and virus infection 48h with Hoechst (1:2000)
Cell plates are placed in high intension cell screening system (PerkinElmer) and observed, using green fluorescent protein fluorescence by cytochrome core
Channel and nuclear targeting fluorescence channel scanning analysis count every hole infection positive cell number and total cell number, calculate virus sense
Dye rate calculates drug to the inhibiting rate of virus replication.
3.2 drugs are to virus replication inhibitory effect
ZIKV-GFP infects BHK-DR cell and after drug effect, in high intension cell screening system
(PerkinElmer) it observes and records and analyzes as a result, antiviral drugs Ribavirin has inhibition to zika virus as the result is shown
Effect, less than 5 μM, fluorescent image is shown when drug concentration is 20 μM, almost without fluorescence signal, when drug concentration is low IC50
When 5 μM, virus infection positive cell rate and control group are reduced than significant difference, and Ribavirin is right under these types of concentration
Cell is without overt toxicity (Fig. 8 A-C).
Drug 6-Azauridine is inhibited to flavivirus such as WNV in pervious report, in nearest report
In it is also inhibited to ZIKV, our test result again shows that the drug has ZIKV-GFP and apparent inhibits effect
Fruit.Concentration is the viral infection rate that can significantly reduce cell after 2 μM of drug-treated cell, drug to the toxicity of cell also compared with
Small, only when concentration is 32 μM, just display significantly affects (Fig. 9 A-C) to cell activity.
Test result shows that Chloroquine also has obvious inhibiting effect to ZIKV-GFP.When drug concentration is 4 μM
Viral infection rate can be significantly reduced.When drug concentration is 16 μM, 80% or more can be reduced to viral infection rate, and drug is dense
At 2-16 μM, display has no significant effect (Figure 10 A-C) to cell activity to degree.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture is (in China Animal Health and Epidemiology Center Harbin point
The heart)
<120>a kind of medicament sifting motion system of recombination zika virus based on expressing green fluorescent protein and its application
<130> KLPI190266
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1218
<212> DNA
<213> DC-SIGNR
<400> 1
gagctcgcca ccatgagtga ctccaaggaa ccaagggtgc agcagctggg cctcctggaa 60
gaagatccaa caaccagtgg catcagactt tttccaagag actttcaatt ccagcagata 120
catggccaca agagctctac agggtgtctt ggccatggcg ccctggtgct gcaactcctc 180
tccttcatgc tcttggctgg ggtcctggtg gccatccttg tccaagtgtc caaggtcccc 240
agctccctaa gtcaggaaca atccgagcaa gacgcaatct accagaacct gacccagctt 300
aaagctgcag tgggtgagct ctcagagaaa tccaagctgc aggagatcta ccaggagctg 360
acccagctga aggctgcagt gggtgagttg ccagagaaat ccaagctgca ggagatctac 420
caggagctga cccggctgaa ggctgcagtg ggtgagttgc cagagaaatc caagctgcag 480
gagatctacc aggagctgac ccggctgaag gctgcagtgg gtgagttgcc agagaaatcc 540
aagctgcagg agatctacca ggagctgacc cggctgaagg ctgcagtggg tgagttgcca 600
gagaaatcca agctgcagga gatctaccag gagctgacgg agctgaaggc tgcagtgggt 660
gagttgccag agaaatccaa gctgcaggag atctaccagg agctgaccca gctgaaggct 720
gcagtgggtg agttgccaga ccagtccaag cagcagcaaa tctatcaaga actgaccgat 780
ttgaagactg catttgaacg cctgtgccgc cactgtccca aggactggac attcttccaa 840
ggaaactgtt acttcatgtc taactcccag cggaactggc acgactccgt caccgcctgc 900
caggaagtga gggcccagct cgtcgtaatc aaaactgctg aggagcagaa cttcctacag 960
ctgcagactt ccaggagtaa ccgcttctcc tggatgggac tttcagacct aaatcaggaa 1020
ggcacgtggc aatgggtgga cggctcacct ctgtcaccca gcttccagcg gtactggaac 1080
agtggagaac ccaacaatag cgggaatgaa gactgtgcgg aatttagtgg cagtggctgg 1140
aacgacaatc gatgtgacgt tgacaattac tggatctgca aaaagcccgc agcctgcttc 1200
agagacgaat agctcgag 1218
Claims (10)
1. the drug screening of recombination zika virus (Zika virus, ZIKV) of the one kind based on expressing green fluorescent protein (GFP)
System, which is characterized in that the medicament sifting motion system by expressing green fluorescent protein recombination zika virus and expression DC-
The BHK-21 cells of SIGNR form.
2. medicament sifting motion system as described in claim 1, which is characterized in that the recombination stockaded village of the expressing green fluorescent protein
Card virus is prepared by the following method to obtain:
1) according to zika virus gene order, other genes in addition to structural protein gene are synthesized by segmented genes synthetic technology
The GFP reporter sequences gene of the structural protein gene position of group sequence and insertion and deletion, passes through polyclone enzyme enzyme site
It is connected into mammalian expression vector, obtains replicon recombinant plasmid, the open reading frame of the replicon recombinant plasmid is that " CMV is opened
Mover-restriction enzyme site -5 ' UTR- coding 20 amino acid of C protein N-terminal nucleotide sequence-GFP-FMDV2A- encodes E protein C
Hold nucleotide sequence-NS1~5 (non-structural protein 1~5 gene orders) -3 ' UTR-HDVr (fourth hepatovirus core of 22 amino acid
Enzyme gene sequence)-restriction enzyme site "；
2) according to zika virus gene order, structural protein gene sequence and GFP sequence are respectively synthesized by gene synthesis technology
Column, the order of connection of each element are the nucleotide sequence-GFP- of 5 ' UTR-coding ZIKV C protein C-terminal, 38 amino acid
The segment is cloned into lactation by polyclone enzyme enzyme site by 1-134 nucleotide sequences of FMDV2A-C-prM-E-NS1 gene
On animal expression vector, the plasmid containing ZIKV structural gene and green fluorescent protein is obtained；
3) rescue of report type ZIKV
Using the obtained plasmid of step 2) as template, PCR amplification is carried out, amplification, which obtains, contains CMV promoter and 5 ' UTR-volume
1-134 nucleotide of nucleotide sequence-GFP-FMDV2A-C-prM-E-NS1 gene of code ZIKV C protein 38 amino acid of C-terminal
The PCR product of sequence, PCR product are used to report the rescue of type virus；
The rescue of report type ZIKV: HEK-293T cell in 24 orifice plates in using 10%v/v FBS DMEM culture medium culture to density
For 70%-90%, using TransIT-293 Transfection Reagent transfection reagent by obtained PCR product and linear
The replicon recombinant plasmid of change is transfected respectively, after transfecting 72h, is harvested cell and supernatant and is saved in -70 DEG C, obtain expressing green
The recombination zika virus of color fluorescin.
3. medicament sifting motion system as claimed in claim 2, which is characterized in that the zika virus gene order is 2015
The gene order of Brazilian popular ZIKV strain, Genbank accession number are as follows: KX280026.
4. medicament sifting motion system as claimed in claim 2, which is characterized in that be by polyclone enzyme enzyme site in step 1)
Sac I and Not I are connected into mammalian expression vector pCI-neo, obtain replicon recombinant plasmid, which opens
Put the nucleotide sequence-GFP- that reading frame is followed successively by CMV promoter-Sac I-5 ' UTR- coding 20 amino acid of C protein N-terminal
Nucleotide sequence-NS1~5 (non-structural protein 1~5 gene orders) -3 of FMDV2A- coding 22 amino acid of E protein C-terminal '
UTR-HDVr (fourth hepatovirus ribozyme gene sequence)-Not I.
5. medicament sifting motion system as claimed in claim 2, which is characterized in that be by polyclone enzyme enzyme site in step 2)
XhoI and NotI is by the nucleotide sequence-GFP-FMDV2A-C-prM-E- of 5 ' UTR-coding ZIKV C protein 38 amino acid of C-terminal
1-134 nucleotide sequences of NS1 gene are cloned on pCI-neo carrier.
6. medicament sifting motion system as claimed in claim 2, which is characterized in that step 3) is using the obtained plasmid of step 2) as mould
Plate, with primer P1:5 '-GGCCTTTTGCTCACATGGCTCGACAG-3 ' and P2:5 ' GACTGCTGCTGCCAATCTACGGGGG-
3 ' carry out PCR amplification, and PCR product is used to report the rescue of type virus.
7. medicament sifting motion system as described in claim 1, which is characterized in that the BHK-21 cell of the expression DC-SIGNR
System constructs obtain by the following method:
1) expression plasmid containing DC-SIGNR is constructed；
2) the DC-SIGNR expression plasmid prepared is linearized, after glue recycles, BHK-21 cell is transfected；
3) screening obtains the BHK-21 cells for stablizing expression DC-SIGNR.
8. medicament sifting motion system as claimed in claim 7, which is characterized in that the coding nucleotide sequence of DC-SIGNR such as SEQ
Shown in ID NO.1, the DC-SIGNR sequence after synthesis is cloned into pCAGneo carrier.
9. the described in any item medicament sifting motion systems of claim 1-8 are screening the application in anti-zika virus drug.
10. a kind of method for screening anti-zika virus drug using the described in any item medicament sifting motion systems of claim 1-8,
It is characterized in that, comprising the following steps:
1) drug-treated and virus infection: the BHK-21 cells for expressing DC-SIGNR are connect with 2%v/vFBS100ul culture medium
Kind in 96 orifice plate of black, when cell density is up to 90%, diluted with the DMEM culture medium of 50ul FBS containing 2%v/v to be measured
The recombination zika virus infection of expressing green fluorescent protein, in 37 DEG C, 5%CO are added after drug effect 1h, 1h2It is trained in incubator
Support 48h；
2) high intension system statistical analysis: cell, will with Hoechst staining cell core after drug-treated and virus infection 48h
Cell plates are placed in high intension cell screening system (PerkinElmer) observation, using green fluorescent protein fluorescence channel and cell
Nuclear staining fluorescence channel scanning analysis counts every hole infection positive cell number and total cell number, calculates viral infection rate, calculate to
Drug is surveyed to the inhibiting rate of virus replication.
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