CN110468145A - A kind of EV-D68 type pseudovirus and its packing method - Google Patents
A kind of EV-D68 type pseudovirus and its packing method Download PDFInfo
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Abstract
The present invention discloses a kind of expressor plasmid packed for packing EV-D68 type pseudovirus, the expressor plasmid of the EV-D68 type pseudovirus is named as D68-EGFP-CAPSID plasmid, and the recombinant expression plasmid has nucleotide sequence shown in SEQ ID NO:1 in sequence table；A kind of for packing the replicon plasmid of EV-D68 type pseudovirus, the replicon plasmid of the EV-D68 type pseudovirus is named as D68-LUC-REPLICON plasmid, and the replicon plasmid has nucleotide sequence shown in SEQ ID NO:2 in sequence table.Further, the plasmid contains D68 virus 5 ' UTR, P2-P3-3 ' UTR gene and firefly luciferase reporter gene (Luciferase) gene.A kind of EV-D68 type pseudovirus of the present invention and its packing method successfully pack out the D68 pseudovirus of single cycle infection, the pseudovirus can be used to substitute D68 live virus, without using safety problem when live virus, the a variety of researchs that can be applied to D68 virus have important application value to the basic research of D68 virus.
Description
Technical field
The present invention relates to field of biotechnology, specially a kind of EV-D68 type pseudovirus and its packing method.
Background technique
68 type of enterovirus D group (Enterovirus D68, EV-D68) belongs to Human enterovirus virus's D group, original name rhinovirus
87 types, for no 20 face cubic symmetry spheric granules of coating, size 23-30nm includes the sub-thread of 7500 nucleotide of treaty
Positive chain RNA.Nucleic acid includes an open reading frame, a poly precursor protein is encoded, before being further hydrolyzed to P1-P3 tri-
Body protein can form tetra- viral capsid proteins of VP1-VP4 after the cutting of P1 precursor protein, P2 and P3 precursor protein can be formed 2A,
Seven functional proteins of 2B, 2C, 3A, 3B, 3C, 3D.D68 is separated and is identified for the first time in the infantile pneumonia in the U.S. in 1962.
In August, 2014, D68 virus come into vogue from Middle West area and then spread to each state in the whole nation, and 49, the whole America state is shared
1153 people infect D68, and up to 14, mostly infant, D68's death for becoming unprecedented is very popular in patient diagnosed.It
There is D68 infection report in countries and regions such as America, Europe, Asia, Africa afterwards.D68 infection symptoms and other enteroviruses
Difference, D68 lead to acute respiratory infections, more seriously multiple studies have shown that D68 infection will lead to acute flaccid ridge
Marrow inflammation (Acute Flaccid Myelitis, AMF), causes irreversibility neurotrosis to the infected.D68 may also lead to adult
Infection, therefore D68 live virus is operated with certain bio-safety hidden danger.The basic research in relation to D68 is weak at present, epidemiology
Investigate less, and these researchs generally need to operate D68 live virus, and operate D68 live virus with certain bio-safety hidden danger,
Therefore the D68 pseudovirus that packaging provides biological safety is of great significance.
Pseudovirus refers to that a kind of retrovirus can integrate the membrane glycoprotein of another variety classes virus, is formed
The cyst membrane with exogenous virus, and genome remains the virus of retrovirus genomic characterization itself.Pseudovirus by
In nucleic acid molecules defective and only one cell infection period, therefore there is higher biological safety, this has to grinding to make internal disorder or usurp
Pathogenicity rate is high, and infectiousness is strong, virus such as HIV, SARS for being not easy to cultivate in vitro provide one kind and safely and effectively grind the side of making internal disorder or usurp
Method.The pseudovirus of external structure also has many advantages, such as that stability is strong, host's preferendum is wide, and it is thin to be therefore widely used in virus host
The screening of born of the same parents, the research and development of vaccine, Neutralization and crystallization research etc..
Therefore, important meaning will be had by successfully constructing a kind of EV-D68 pseudovirus.
Summary of the invention
To solve above existing issue, the present invention provides a kind of EV-D68 type pseudovirus and its packing method.The present invention is logical
Cross following technical scheme realization.
It is a kind of for packing the expressor plasmid of EV-D68 type pseudovirus, the expressor plasmid of the EV-D68 type pseudovirus
It is named as D68-EGFP-CAPSID plasmid, the recombinant expression plasmid has nucleotide shown in SEQ ID NO:1 in sequence table
Sequence.
Further, the recombinant plasmid uses Green fluorescent protein fusion vector (EGFP gene), and the plasmid contains
D68 virus structural protein gene (P1 gene)；Between the EGFP gene and P1 gene be inserted into 2A proteolytic cleavage site (-
NIVTT-)。
Further, a method of expression D68-EGFP-CAPSID plasmid comprising the steps of: 1) green fluorescence
The gene order of albumen and the gene order of all structural proteins of D68 are spliced；2) and by the restriction enzyme site of 2A protease it is inserted into it
Between；3) design primer；
The primer sequence are as follows:
D68-EGFP-F gggagACCCAAGCTGGCTAg
D68-EGFP-R CTAGTAACTTGAGCCCCCATaagggtagtaatggccttgtaCAGc
D68-P1-F acaaggccattactacccttATGGGGGCTCAAGTTACTAGACAAC
D68-P1-R tgatggtgatgatgaccggtttaATTGGTCACTAACCTGATATCATGAGGc。
It is a kind of for packing the replicon plasmid of EV-D68 type pseudovirus, the replicon plasmid of the EV-D68 type pseudovirus
It is named as D68-LUC-REPLICON plasmid, the replicon plasmid has nucleotide shown in SEQ ID NO:2 in sequence table
Sequence.
Further, the plasmid contains D68 virus 5 ' UTR, P2-P3-3 ' UTR gene and firefly luciferase report
Gene (Luciferase) gene.
Further, plasmid firefly luciferase reporter gene (Luciferase) gene and P2-P3-3 '
2A proteolytic cleavage site (- NIVTT-) is inserted between UTR gene.
A kind of construction method of D68-LUC-REPLICON plasmid comprising the steps of: 1) amplification obtain 5 ' UTR genes,
Luciferase gene, P2-P3-3 ' UTR gene；2) and by the restriction enzyme site of 2A protease insertion Luciferase gene and
Between P2-P3-3 ' UTR gene；3) three above genetic fragment is seamlessly connected；4) design primer；
The primer sequence are as follows:
D68-5UTR-F cgtcttcaagaattgcggccgcgtaatacgactcactataggTTAAAACAGCCT
TGGGGTTGTTCC
D68-5UTR-R atgtttttggcgtcttccatCTTTGTGAACAGGTTTAGAATCTTCAAAA
D68-LUC-F GTTCACAAAGatggaagacgccaaaaacataaagaaag
D68-LUC-R ccATTGGTCACTAACCTGATcaatttggactttccgcccttc
D68-2A-3UTR-F gaaagtccaaattgATCAGGTTAGTGACCAATGGC
D68-2A-3UTR-R tcaaacatgagaattgtcgacTTTTTTTTTTTTTTTTTTTTTTTTT
Overlap-F cgtcttcaagaattgcggcc
Overlap-R tcaaacatgagaattgtcgacTTTTTTTTTTTTTTTTTTTTTTTTT
A kind of EV-D68 type pseudovirus, by the D68 structural proteins of the D68-EGFP-CAPSID plasmid expression and described
The D68 subgenomic RNA of D68-LUC-REPLICON replicon transcription assembles.
A kind of packing method of EV-D68 type pseudovirus, be by D68-EGFP-CAPSID, D68-LUC-REPLICON and
PcDNA-T7-polymerase cotransfection 293T cell, then harvests pseudovirus.
Beneficial effects of the present invention:
The present invention constructs the expressor plasmid of EV-D68 type pseudovirus, replicon plasmid is used to pack D68 pseudovirus, success
The D68 pseudovirus of single cycle infection is packed out, when which can be used to substitute D68 live virus, do not have using live virus
Safety problem, can be applied to a variety of researchs of D68 virus, have important application value to D68 basic research.
Detailed description of the invention
Fig. 1 is enterovirus genome schematic diagram；
Fig. 2 is single cycle infection pseudovirus schematic diagram；
Fig. 3 is the gene magnification of D68 expressor and identification electrophoretogram；
Fig. 4 is D68 replicon gene fragment amplification and seamless connection fragment electrophoretic figure；
Fig. 5 is D68 replicon identified for genes electrophoretogram；
Fig. 6 is the qualification figure of EV-D68 type pseudovirus on a molecular scale；
Fig. 7 is qualification figure of the EV-D68 type pseudovirus in protein level；
Fig. 8 is EV-D68 type pseudovirus viral copy number result；
Fig. 9 is EV-D68 type pseudovirus infection ability result.
Specific embodiment
Technical solution of the present invention work specifically, is completely illustrated with reference to the accompanying drawing.Used in embodiment
Experimental method is conventional method unless otherwise specified.Material, reagent used etc. are unless otherwise specified commercially available.
1. experimental material: RD cell (deriving from human rhabdomyosarcoma) (being purchased from ATCC) and 293T cell (derive from people's embryo
Foetus renal cells) (be purchased from ATCC) cultivate in the DMEM of 10%FBS (fetal calf serum is purchased from GIBCO).RD cell is for detecting
The sensitive cell line of neutralizing antibody test.293T cell is used to the pseudovirus of packaging CA10.Enterovirus D group 68 type virus
BCH895A plants, Chinese epidemic strain, GenBank:KF726085.1 is purchased from ATCC.
2. 68 type expressor plasmid construction method of enterovirus D group:
Using the domestic epidemic strain of enterovirus D group 68 type virus saved in laboratory, this department, extracted by viral RNA
Kit obtains the full-length genome (shown in Fig. 1) of D68 Strain, and passes through RT-PCR (Reverse Transcription
PCR the capsid protein expressing gene P1 of D68) is expanded；And the expression vector CA6-EGFP- containing EGFP gene saved with department
PcDNA6.0 is template, and the P1 segment and EGFP segment for being able to carry out seamless connection, in detail specificity nothing are obtained by PCR amplification
Seam connection primer table, in design of primers, EGFP reverse primer and the forward primer of P1 segment add 2A restriction enzyme site sequence
(-NIVTT-)；Meanwhile by restriction enzyme A geI and NheI, double enzymes are carried out to eukaryotic expression vector pcDNA6.0
It cuts (shown in Fig. 2).PcDNA6.0 carrier after the P1 segment of PCR amplification acquisition and EGFP segment and double digestion is carried out together seamless
Connection reaction.The product of acquisition is converted, picking monoclonal colonies, extracts plasmid.With AgeI and NheI double digestion and
Plasmid after the connection of AgeI single endonuclease digestion carries out Preliminary Identification, as a result sees that Fig. 3 is shown in, the band molecular size range after digestion meets pre-
Phase, and sequencing result shows that matching rate is 100%, illustrates that capsid protein expression vector D68-EGFP-CAPSID building is correct.It will
The D68 cape horn fever virus capsid protein expression vector D68-EGFP-P1 transfection 293T cell built, due to will be glimmering in vector construction
Photoprotein EGFP and P1 amalgamation and expression, therefore the brightness of EGFP can be observed by fluorescence inverted microscope to judge the expression of P1
Situation.Positive control by only expressing EGFP carrier with transfection is compared, it may be determined that the capsid protein expression vector of building
It being capable of normal expression.The primer is as follows:
3. 68 type replicon plasmid construction method of enterovirus D group:
D68 cape horn fever replicons Replicon is constructed based on the full-length genome cDNA of the domestic popular strain virus of D68
Made of.The full-length genome of D68 Strain is obtained by viral RNA extraction agent box, and passes through RT-PCR (Reverse
Transcription PCR) segmentation amplification gene segment, amplified fragments are divided into 5 ' UTR (710bp), P2-P3-3 ' UTR
Two sections of (4078bp) carries out RT-PCR amplification by the primer that specificity can be seamlessly connected, obtains 2 two molecular weight therewith
The specific band that size is consistent, is shown in Fig. 4.And the expression vector of the gene containing Luciferase saved with department
EV71Luciferase-PSVA is template, and the Luciferase segment for being able to carry out seamless connection is obtained by PCR amplification.It is logical
Lap over PCR (Overlap PCR) is reacted 5 ' UTR, Luciferase, D68 Strain structural gene P2-P3-3 ' of acquisition
Tri- segment connections of UTR.PCR fragment is building up on PSVA carrier according to design diagram by seamless connection reaction, and is led to
Cross primer addition T7 promoter sequence (gtaatacgactcactatagg) with guarantee RNA polymerase identification combine transcription and
PolyA sequence is to guarantee the stability of mRNA.The product of seamless connection is converted, picking monoclonal colonies, extracts plasmid.
The plasmid of acquisition is first subjected to double digestion with NotI and SalI and carries out Preliminary Identification, sees Fig. 5.Fragments molecules amount size as the result is shown
Meet expection, correct carrier will be constructed and verified by sequencing, the final D68- for obtaining sequences match rate 100%
Replicon expression vector.D68-Replicon will be obtained and t7 rna polymerase expression plasmid transfects 293T cell jointly, screened
Suitable transfection reagent and transfection amount, and whether replicon carrier building is judged by the fluorescent value RLU of detection Luciferase
It is correct and can normal expression, test result shows the fluorescent value RLU of Luciferase expression up to 105, it was demonstrated that D68-
Replicon building is correct and being capable of normal expression.
3. transfection and pseudovirus packaging
Transfection first 12-14 hours in 10cm2Tissue culture plate upper berth 293T cell.The transfection same day, the density of 293T are answered
This reaches 80-90%.Previous hour is transfected, cultivates 293T cell with 10%FBS-DMEM.With (purchased from Polyplus, product mesh
Record number is 114-15) transfection 10 μ g tri- kinds of plasmid mixture Ds 68-EGFP-CAPSID, D68-LUC-REPLICON, pcDNA-T7-
Polymerase (mass mixings such as three kinds of plasmids), is cultivated in 37 DEG C of cell incubators.In fluorescence microscopy microscopic observation after for 24 hours
EGFP fluorescin is to judge the expression of structural proteins.After 48h, cell conditioned medium is collected into 15ml pipe, in cell
Surface leaves the culture medium of about 500 μ l.293T cell is resuspended with remaining culture medium, and with dry ice fast freeze-thaw two repeatedly
It is secondary.The precipitating for the culture medium supernatant freeze thawing collected before is mixed, then uses centrifuge 2 again, 500g is centrifuged 15 minutes, is abandoned
Precipitating is collected supernatant, and is dispensed into EP pipe I.5ml, and it is spare to be stored in -80 DEG C of refrigerators.
The identification of 4.EV-D68 type pseudovirus
For identification EV-D68 type pseudovirus on a molecular scale, by viral RNA extraction agent box to D68 pseudovirus
Harvest liquid and cell harvest liquid carry out the extraction of genome, the full-length genome of pseudovirus and cell sample are obtained, with the full genome
Group is that template carries out RT-PCR (Reverse Transcription PCR) amplification Luciferase genetic fragment.Experimental result
As shown in Figure 6.Pseudovirus sample can detect Luciferase gene and molecular size range is consistent with positive control, illustrate to pack
Pseudovirus in contain Luciderase gene, that is, contain replicon Replicon-RNA in the pseudovirus harvested；In molecular water
Illustrate that the building of pseudovirus is correct on flat.
In order to identify that EV-D68 type pseudovirus in the characteristic of protein level, transfects cell precipitation as sample using D68 pseudovirus
Product carry out protein blot test (Western Blot).Wherein positive control is D68 live virus infection cell precipitating, negative control
For the cell sample of collection；For identifying that the primary antibody of D68 pseudovirus is the source of mouse polyclonal antibody for D68 live virus.As a result
Referring to Fig. 7, the results showed that, D68 live virus infection cell precipitating is as positive control (swimming lane 2) it can be observed that capsid protein
VP1 (32kDa) band, this plays main antigenic with VP1 in D68 virus and matches；Pseudovirus sample (swimming lane 1) can be seen
Apparent VP1 band is observed, molecular size range matches with positive control size substantially.The test proves on protein level
The pseudovirus of packaging has certain antigenicity.
In order to detect the copy number of EV-D68 type pseudovirus virus, pseudovirus leads to it due to the imperfection of its genome
To only one infectious cycle of cell, will not occur apparent cytopathy as D68 live virus, therefore in virus titer knot
When the judgement of fruit, the infection ability of observation cytopathy situation judgement virus cannot be passed through.Therefore, it is obtained to evaluate packaging
D68 pseudovirus titre, the finger using the copy number of real-time quantitative RT-PCR measurement pseudovirus sample, as evaluation pseudovirus titre
Mark.As a result see Fig. 8, measure three as the result is shown and permit a leave virus titer result as 4-8 × 106copies/ml.Due to packing vacation every time
When viral, the pseudovirus titre that difference will lead to different batches between cell state and batch can be changed, therefore harvested every time
Its specific virus titer is detected after pseudovirus, so as to follow-up test operation.
It is dilute according to 2 times of series by the way that successful D68 pseudovirus will be packed in order to detect EV-D68 type pseudovirus infection ability
It releases, infects RD cell respectively, culture medium does negative control, is placed in 33 DEG C of incubators and cultivates, and supernatant is abandoned after culture and is used in combination
Culture medium gently rinses cell surface 2 times, and 1 × cell pyrolysis liquid is added, and part lysate is transferred to 96 afterwards twice by multigelation
Hole white detection plate.The chemiluminescence of RD cell is infected by the pseudovirus under chemiluminescence imaging system detection difference dilution
Signal strength (relative light unit, RLU).Draw fluorescence intensity RLU and pseudovirus extension rate histogram.Referring to
Fig. 9, D68 pseudovirus can detecte fluorescence signal as the result is shown, and also therewith with the dilution fluorescence signal value of pseudovirus
It reduces, illustrates that D68 pseudovirus has infection ability.
To sum up the experimental results showed that, the D68 pseudovirus of packaging has with provirus similar characteristic.
D68 pseudovirus is successfully established the flow of research for having pushed us to 68 type of enterovirus D group, to develop in the future
The prevention and treatment vaccine of D68 virus, or find suitable effective drug screening and exploitation provide it is a kind of safely, effectively,
Practical tool.
The foregoing is merely the preferable specific embodiments of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art's change or replacement obtained without making creative work, are all answered
It is included within the scope of the present invention.
Claims (9)
1. a kind of for packing the expressor plasmid of EV-D68 type pseudovirus, it is characterised in that: the EV-D68 type pseudovirus
Expressor plasmid is named as D68-EGFP-CAPSID plasmid, and the recombinant expression plasmid has SEQ ID NO:1 institute in sequence table
The nucleotide sequence shown.
2. recombinant expression plasmid according to claim 1, it is characterised in that: the recombinant plasmid uses green fluorescent protein
Reporter gene (EGFP gene), the plasmid contain D68 virus structural protein gene (P1 gene)；The EGFP gene and P1 base
Therefore 2A proteolytic cleavage site (- NIVTT-) is inserted between.
3. the construction method of recombinant expression plasmid according to claim 1, it is characterised in that: comprise the steps of: 1) green
The gene order of color fluorescin and the gene order of all structural proteins of D68 are spliced；2) and by the restriction enzyme site of 2A protease
Between insertion；3) design primer；
The primer sequence are as follows:
D68-EGFP-F gggagACCCAAGCTGGCTAg
D68-EGFP-R CTAGTAACTTGAGCCCCCATaagggtagtaatggccttgtaCAGc
D68-P1-F acaaggccattactacccttATGGGGGCTCAAGTTACTAGACAAC
D68-P1-R tgatggtgatgatgaccggtttaATTGGTCACTAACCTGATATCATGAGGc。
4. a kind of for packing the replicon plasmid of EV-D68 type pseudovirus, which is characterized in that the EV-D68 type pseudovirus
Replicon plasmid is named as D68-LUC-REPLICON plasmid, and the replicon plasmid has SEQ ID NO:2 institute in sequence table
The nucleotide sequence shown.
5. a kind of replicon plasmid according to claim 4, it is characterised in that: the plasmid contains D68 virus 5 ' UTR,
P2-P3-3 ' UTR gene and firefly luciferase reporter gene (Luciferase) gene.
6. a kind of replicon plasmid according to claim 4, it is characterised in that: the plasmid firefly luciferase report
2A proteolytic cleavage site (- NIVTT-) is inserted between gene (Luciferase) gene and P2-P3-3 ' UTR gene.
7. the construction method of replicon plasmid according to claim 4, it is characterised in that: comprise the steps of: and 1) expand
Obtain 5 ' UTR genes, Luciferase gene, P2-P3-3 ' UTR gene；2) it and by the restriction enzyme site of 2A protease is inserted into
Between Luciferase gene and P2-P3-3 ' UTR gene；3) three above genetic fragment is seamlessly connected；4) design is drawn
Object；
The primer sequence are as follows:
D68-5UTR-F cgtcttcaagaattgcggccgcgtaatacgactcactataggTTAAAACAGCCTTGG
GGTTGTTCC
D68-5UTR-R atgtttttggcgtcttccatCTTTGTGAACAGGTTTAGAATCTTCAAAA
D68-LUC-F GTTCACAAAGatggaagacgccaaaaacataaagaaag
D68-LUC-R ccATTGGTCACTAACCTGATcaatttggactttccgcccttc
D68-2A-3UTR-F gaaagtccaaattgATCAGGTTAGTGACCAATGGC
D68-2A-3UTR-R tcaaacatgagaattgtcgacTTTTTTTTTTTTTTTTTTTTTTTTT
Overlap-F cgtcttcaagaattgcggcc
Overlap-R tcaaacatgagaattgtcgacTTTTTTTTTTTTTTTTTTTTTTTTT。
8. a kind of EV-D68 type pseudovirus, it is characterised in that: by the D68 structure egg of the D68-EGFP-CAPSID plasmid expression
The D68 subgenomic RNA of the white and described D68-LUC-REPLICON replicon transcription assembles.
9. a kind of packing method of pseudovirus described in claim 8, it is characterised in that: be by D68-EGFP-CAPSID, D68-
LUC-REPLICON and pcDNA-T7-polymerase cotransfection 293T cell, then harvests pseudovirus.
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Cited By (2)
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| CN108424881A (en) * | 2018-03-23 | 2018-08-21 | 中国食品药品检定研究院 | One enterovirus, 68 type and its application in preparing EV-D68 type infection animals |
| CN113201599A (en) * | 2021-06-03 | 2021-08-03 | 北京大学人民医院 | Method for detecting pathogens infected with cerebrospinal fluid based on PCR and nanopore sequencing |
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