CN108359640A - A kind of rabies viruses of the full brain area neural network structure of label, it is prepared and application - Google Patents
A kind of rabies viruses of the full brain area neural network structure of label, it is prepared and application Download PDFInfo
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Abstract
The invention belongs to the structure fields of stable cell lines, and more particularly, to a kind of rabies viruses of the full brain area neural network structure of label, it is prepared and application.The present invention completes the preparation of BHK N2C (G) cell line using third generation slow virus technology, packed by the structure of FUGW H2B GFP P2A N2C (G) slow virus carrier and virus, it infects bhk cell and obtains BHK N2C (G) cell with nuclear location fluorescence, which is used for the production of deficiency recombinant rabies poison.BHK N2C (G) cell is infected with RV △ G X virus liquids, RV N2C (G) △ G X products are prepared by amplification purification.Show recombination deficient mutant rabies viruses RV N2C (G) △ G X prepared by the present invention in injection site inflammatory reaction unobvious by live body test result, reverse projected area is more, and reverse projection efficiency is high.
Description
Technical field
The invention belongs to the structure fields of stable cell lines, more particularly, to a kind of full brain area neural network knot of label
The rabies viruses of structure, it is prepared and application.
Background technology
Cerebral nerve network is made up of Synaptic junction the different neuron of huge number and form, characteristic
Labyrinth is that brain exercises the movable architecture basics such as cognition, emotion, memory, the imagination.Disclose the knot of cerebral nerve network
Structure is the basic premise for the mechanism for finally understanding brain processing information.Traditional neural network tracing method, such as Electronic Speculum, Gao Er
Base dyeing, dyestuff, protein and peptide class marker can show the form of brain area neuron and its throwing to other brain areas
It penetrates, the form of particular cell types neuron in brain can be also shown using transgenic mice；A small number of protein-based tracers are also
Across cynapse label may be implemented, but these tracing methods with signal decay seriously by not special, across the postsynaptic signal in indirect, direction
The problems such as.Above-mentioned tradition tracing method promotes understanding of the people to cerebral nerve network structure, but is difficult to use in research by more
The Complex Neural Network that a brain area, multiple types neuron are formed by Synaptic junction, and this neural network is only at brain
The loop that reason specific information is relied on.Neurotropic virus, which is one kind, can infect nerve cell, and can propagate and rise in value along neural circuitry
Virus.Neurotropic virus labelling technique is tracking cerebral nerve loop structure, parses the important hand of cerebral nerve network connection
Section.
Using means such as reverse genetics and homologous recombinations, on the basis of vaccine strain, viral genome is transformed, knocks out special
Determine virulence gene, be inserted into the foreign genes such as fluorescin, obtains less toxic power, recombination tracer work safe to use, carrying marker
Tool virus.This kind of recombination tracer tool virus still has orientation across cynapse and replication capacity, suitable label neural network structure.
Wherein, RV (Rabies virus, RABV) is recombinated because its retrograde labeled ability is widely used in loop tracer.
RV is the rabic virulence factor of infecting both domestic animals and human, can be infected including all kinds of wild animals.Wild type RV has
The characteristic inversely propagated across cynapse, can be used for primate and the neuroanatomical research of rodent.Tracer is done using RV
Tool virus has unique advantage, in addition to across the cynapse propagation of specificity, after RV infects cental system, and main mark neuron, almost
Do not mark spongiocyte；The nerve fibre of not Synaptic junction is not infected, toxicity is low, and infected neuron hardly happens
Apparent lesion and cracking.
The prior art, which lacks, clearly the fine form of labeled neurons and efficiently inversely to mark other brains
The RV virus systems in area, and there is the big defect of injection site inflammatory reaction in the living body in the virus of prior art production.
Invention content
For the disadvantages described above or Improvement requirement of the prior art, the present invention provides a kind of full brain area neural network knots of label
The rabies viruses of structure, it is prepared and application, by providing a kind of cell line that can stablize generation envelope glycoprotein N2C, and
With cell line production deficiency recombinant rabies poison, virus yield is high, by the deficiency recombinant rabies poison for marking god
It is tested through network live body, live body test shows in injection site inflammatory reaction unobvious, and the reverse brain area that projects is more, reverse to project
It is more efficient.
To achieve the above object, according to one aspect of the present invention, a kind of envelope glycoprotein expression cell system is provided,
For bhk cell, the cell line includes envelope glycoprotein gene N2C (G), and the virus of envelope glycoprotein packaging can be from nerve
First axon ends inversely absorb infection nerve cell；The cell line can express N2C (G) albumen, and can stablize passage 50 generations with
On.
Preferably, the envelope glycoprotein N2C is the envelope glycoprotein of Strain CVS.
Preferably, the cell line is built to obtain by following methods：
(1) H2B-GFP-P2A-N2C (G) segment, gene order such as SEQ ID NO are obtained by PCR amplification：1 institute
Show；Using slow virus FUGW as carrier, promoter downstream GFP genes replace with H2B-GFP-P2A-N2C (G), build core plasmid
FUGW-H2B-GFP-P2A-N2C(G)；
(2) by the plasmid transfection 293T cells, supernatant filtering is collected in 48h~120h, obtains FUGW-H2B-GFP-
P2A-N2C (G) slow virus；
(3) by the FUGW-H2B-GFP-P2A-N2C being collected into (G) slow-virus infection bhk cell, infection multiplicity MOI is 5,
Cell state and Fluorescence Ratio are observed after 48h~120h and is passed on, and Fluorescence Ratio reaches 90% or more, after passing on 5~7 times
Conservation is carried out, BHK-N2C (G) cell is obtained.
Other side according to the invention provides a kind of application of the cell line, is used to prepare neural markers
Preparation.
Preferably, the cell line is used to prepare full brain area neural markers preparation.
Other side according to the invention provides a kind of rabies viruses for marking brain area nerve, is SAD-
B19 attenuated strains, viral capsid contain envelope glycoprotein N2C (G), and envelope glycoprotein N2C (G) is the coating of Strain CVS
Glycoprotein.
Other side according to the invention provides a kind of preparation method of the rabies viruses, including walks as follows
Suddenly：
(1) it waits for that the passage of BHK-N2C (G) cell is stablized, 0.05% or 0.25% pancreatin of cell is digested into 2~3min, is pressed
According to 1:5~1:7 ratio is passed on, and is cultivated in 35~37 DEG C of incubators；
(2) 4-6h is cultivated, culture medium in culture bottle is abandoned, RV- △ G-dsred diseases of the 13~15mL through freeze thawing and filtering is added
Poison is infected, and is transferred in 33~35 DEG C of incubators and is continued to cultivate；
(3) 48~72h is infected, when infection proportion reaches 80% or more, virocyte is subjected to digest amplification, 35~37 DEG C
Culture in incubator；
(4) it waits for that cell density reaches 70%-80%, cell is transferred in 33~35 DEG C of DEG C of incubators and is cultivated, and collects disease
Venom supernatant simultaneously adds fresh culture, and virus liquid supernatant is concentrated and purified, the virus is obtained.
Other side according to the invention provides a kind of application of the rabies viruses, is used to prepare neural mark
Remember preparation.
Preferably, the rabies viruses is used to prepare the label preparation for marking full cranial nerve.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect：
(1) the present invention provides the stable cell lines containing envelope glycoprotein N2C genes, which can stablize expression packet
Membrane glycoprotein N2C；The cell line using slow virus can the stability of characteristics of random integration to host cell gene group express N2C
(G) it is more than generation to stablize passage 50 for albumen.
(2) present invention completes the preparation of BHK-N2C (G) cell line by using third generation slow virus technology, that is, passes through
The structure of FUGW-H2B-GFP-P2A-N2C (G) slow virus carrier is packed with virus, and infection bhk cell obtains band nuclear location fluorescence
BHK-N2C (G) cell, the cell be used for deficiency recombinant rabies poison production, that is, use RV- △ G-X virus liquids infection
RV-N2C (G)-△ G-X products are prepared by amplification purification in BHK-N2C (G) cell, and virus production yield is high.
(3) envelope glycoprotein expression cell provided by the invention system, the defect for the full cranial nerve network of label being prepared
Type recombinant rabies poison, by live body test result show deficiency recombinant rabies poison injection site inflammatory reaction significantly
It reduces, the brain area inversely projected increases, and reverse projection efficiency also higher inversely marks for deficiency rabies viruses in neural network
In application above provide better research tool.
Description of the drawings
Fig. 1 is that core plasmid FUGW-H2B-GFP-P2A-N2C (G) made from the embodiment of the present invention 1 and skeleton plasmid are total
Transfect 293T cells, for 24 hours with fluorescing matter figure after 48h；
Fig. 2 is the cell states and Fluorescence Ratio figure of BHK-N2C (G) stable cell lines；
Fig. 3 is BHK-N2C (G) cytological map of recovery；
Fig. 4 is RV-N2C (G)-△ G-dsred cytological maps that the embodiment of the present invention 1 is prepared；
Fig. 5 is the titre test chart of 1RV-N2C of the embodiment of the present invention (G)-△ G-dsred；
Fig. 6 is 1 injection site figure of the embodiment of the present invention；
Fig. 7 A are that cingulate gyrus cortex cg1 and leading edge cortex Pr1 inversely marks result figure；Fig. 7 B are motor cortex M1+M2
Reverse label result figure；
Fig. 8 is that corpus straitum CPU inversely marks result figure；
Fig. 9 A are that hypothalamic area inversely marks result figure, Fig. 9 B inversely to mark result figure for the areas incertitude zone Z1；
Figure 10 A and Figure 10 B are respectively that the deadlock areas core foot interfascicular fasciculus fr and back seam nucleus DRN inversely mark result figure；
Figure 11 is 1 injection site figure of comparative example；
Figure 12 is the label design sketch that comparative example 1 inversely marks hypothalamus Hypothalamus brain areas；
Figure 13 is the label design sketch that comparative example 1 inversely marks lateral hypothalamus LH brain areas；
Figure 14 is the label design sketch that comparative example 1 inversely marks the areas back seam nucleus DRN.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
It does not constitute a conflict with each other and can be combined with each other.
The present invention provides a kind of envelope glycoprotein expression cell systems, are bhk cell, and the cell line includes rabies
Malicious CVS plants of envelope glycoprotein gene N2C (G), the virus of this envelope glycoprotein packaging is with reverse from neuron axon end
Absorb the characteristic of infection nerve cell.This cell line using slow virus can random integration to host cell gene group stability of characteristics
N2C (G) albumen is expressed, it is more than generation that passage 50 can be stablized.The cell line is built to obtain by following methods：
(1) H2B-GFP-P2A-N2C (G) segment is obtained by PCR amplification, using slow virus FUGW as carrier, under promoter
Trip GFP genes replace with H2B-GFP-P2A-N2C (G), structure core plasmid FUGW-H2B-GFP-P2A-N2C (G)；
(2) by the plasmid transfection 293T cells, supernatant filtering is collected in 48h~120h, obtains FUGW-H2B-GFP-
P2A-N2C (G) slow virus；
(3) by the FUGW-H2B-GFP-P2A-N2C being collected into (G) slow-virus infection bhk cell, infection multiplicity MOI is 5,
Cell state and Fluorescence Ratio are observed after 48h~120h and is passed on, and Fluorescence Ratio reaches 90% or more, after passing on 5~7 times
Conservation is carried out, BHK-N2C (G) cell is obtained.
The cell line can be used for preparing neural markers preparation, for example can be used for preparing a kind of full brain area neural network knot of label
The recombination deficient mutant rabies viruses of structure can prepare the various preparations for marking full brain area nerve using the virus.
Correspondingly, it is SAD- the present invention provides a kind of recombination deficient mutant rabies viruses for marking brain area nerve
B19 attenuated strains, viral capsid contain envelope glycoprotein N2C (G), and envelope glycoprotein N2C (G) is the coating of Strain CVS
Glycoprotein.The preparation method of the recombination deficient mutant rabies viruses, includes the following steps：
(1) it waits for that the passage of BHK-N2C (G) cell is stablized, 0.05% or 0.25% pancreatin of cell is digested into 2~3min,
According to 1:5~1:7 ratio is passed on, and is cultivated in 35~37 DEG C of incubators；
(2) 4~6h is cultivated, culture medium in culture bottle is abandoned, RV- △ G-dsreds of the 13~15mL through freeze thawing and filtering is added
Virus is infected, and is transferred in 33~35 DEG C of incubators and is continued to cultivate；
(3) 48~72h is infected, when infection proportion reaches 80% or more, virocyte is subjected to digest amplification, 35~37 DEG C
Culture in incubator；
(4) it waits for that cell density reaches 70-80%, cell is transferred in 33~35 DEG C of incubators and is cultivated, and collects virus liquid
Supernatant simultaneously adds fresh culture, and virus liquid supernatant is concentrated and purified, the virus is obtained.
For current RV- △ G-X production efficiencys and the inflammatory reaction occurred in neural network retrograde labeled and inverse
The problem of to projection efficiency, the present invention propose it is a kind of to virus target new approaches that relevant envelope glycoprotein is replaced and
New method.The present invention is mainly the preparation that (1) completes BHK-N2C (G) cell line by using third generation slow virus technology, i.e., logical
The structure and virus for crossing FUGW-H2B-GFP-P2A-N2C (G) slow virus carrier are packed, and infection bhk cell obtains band nuclear location
BHK-N2C (G) cell of fluorescence, the cell are used for the production of deficiency recombinant rabies poison.(2) RV- △ G-X virus liquids are used
BHK-N2C (G) cell is infected, RV-N2C (G)-△ G-X products are prepared by amplification purification.(3) live body survey is finally carried out
Examination.Show that recombination deficient mutant rabies viruses RV-N2C (G)-△ G-X prepared by the present invention are injecting position by live body test result
Point inflammatory reaction unobvious, reverse projected area is more, reverse projection efficiency higher.The bright spot of the invention is that：1. improving
The production yields of recombination deficient mutant RV viruses；2. injection site inflammatory reaction in the living body substantially reduces, the brain inversely projected
Area increases, and also higher, recombination deficient mutant rabies viruses RV-N2C (G)-△ G-X prepared by the present invention are one kind to reverse projection efficiency
Excellent full cranial nerve network structure tag preparation above carries for application of the deficiency rabies viruses in neural network inversely marks
Better research tool is supplied.
Embodiment 1
(1) structure of BHK-N2C (G) stable cell lines
(1-1) FUGW-H2B-GFP-P2A-N2C (G) slow virus carrier is built to be packed with virus
FUGW cuts to obtain FUGW (B/E) with BamHI and EcoRI are bis-, with 73476 (addgene numbers) for template, with
H2B-GFP-P2A-N2C (G)-F (same) and H2B-GFP-P2A-N2C (G)-R (same) is primer, 58 degree, 3 minutes of annealing temperature
Amplification obtains H2B-GFP-P2A-N2C (G) segment, gene order such as SEQ ID NO：Shown in 1, FUGW (B/E) and H2B-
GFP-P2A-N2C (G) segment homologous recombination is placed on 37 DEG C of incubator cultures, and bacterium colony PCR identifications are carried out after staying overnight, by positive gram
Grand progress plasmid extraction, digestion verification and sequencing, obtain target plasmid FUGW-H2B-GFP-P2A-N2C (G)；Wherein, H2B is
Histone H2B has and appraises and decides bit function, gene order such as SEQ ID NO：Shown in 2, GFP is green fluorescent protein, gene
Sequence such as SEQ ID NO：Shown in 3, P2A is the small peptide with self splicing function, gene order such as SEQ ID NO：4 institutes
Show；N2C (G) is the envelope glycoprotein of CVS-N2C plants of rabies viruses, gene order such as SEQ ID NO：Shown in 5, in addition, SEQ
ID NO：Added GCCACC is Kozak sequences before H2B genes and GFP genes in 1 sequence.
Correct core plasmid FUGW-H2B-GFP-P2A-N2C (G) and skeleton plasmid cotransfection 293T cells are verified,
For 24 hours with observe fluorescing matter after 48h, and collect supernatant filtering in 48h, obtain the slow diseases of FUGW-H2B-GFP-P2A-N2C (G)
Poison.Figure 1A and Figure 1B is fluorescing matter after transfecting for 24 hours；C, D are fluorescing matter after 48h transfections, it can be seen that Fluorescence Ratio reaches
90% or more, transfection is good, core plasmid normal expression.
The preparation of (1-2) BHK-N2C (G) stable cell lines
The FUGW-H2B-GFP-P2A-N2C being collected into (G) slow virus (being 5 by infection multiplicity MOI) infection BHK is thin
Born of the same parents observation cell state and Fluorescence Ratio and are passed on after 48h, and Fluorescence Ratio reaches 90% or more, is protected after passing on 5 times
Kind.Fig. 2 is the cell states and Fluorescence Ratio figure of BHK-N2C (G) stable cell lines, as can be seen from the figure intracellular green core
Fluorescence Ratio ＞ 90% is positioned, and cell state is good, illustrates that cell line structure is completed.
(2) viral preparation (by taking RV-N2C (G)-△ G-dsred productions as an example)
(2-1) recovery BHK-N2C (G)；Fig. 3 is BHK-N2C (G) cell of recovery.
(2-2) waits for that (general 3-5 generations) is stablized in the passage of BHK-N2C (G) cell, and 0.25% pancreatin of cell is digested 2min,
(general 1 is passed on according to a certain percentage:5-1:7) it, is cultivated in 37 DEG C of incubators.
(2-3) cultivates 4-6h, abandons culture medium in culture bottle, and RV- △ G-dsred diseases of the 15mL through freeze thawing and filtering is added
Poison, and be transferred in 35 DEG C of incubators and continue to cultivate.
(2-4) infects 48-72h, and observation infection conditions press virocyte when nonspecific infection ratio reaches 80% or more
Digest amplification is carried out according to certain proportion, is cultivated in 35 DEG C of incubators.
Cell is transferred in 33 DEG C of incubators and cultivates, and collect virus liquid when cell density reaches 80% or so by (2-5)
Supernatant simultaneously adds fresh culture.Fig. 4 is RV-N2C (G)-△ G-dsred cytological maps, it can be seen that virus is at BHK-N2C (G)
It can be expanded on cell, and amplification rate is very fast, red fluorescence ratio is higher.
(2-6) concentrates and purifies virus liquid supernatant when supernatant reaches certain volume, is used in combination gradient dilution method to dense
Contracting virus titer is measured, and measurement cell is 293T cells.Fig. 5 is the titre test chart of RV-N2C (G)-△ G-dsred,
It can be seen that virus can infect 293T cells, and virus titre after concentrating and purifying increases, and reaches certain amount grade.
(3) viral live body test
Live bodies of RV-N2C (the G)-△ G-dsred that step (2) is prepared for mouse C57 is tested, injection site
For VTA (ventral tegmental area), viral dose 400nL.
Fig. 6 is the label design sketch of injection site figure, Fig. 7 to Figure 10 and reverse label Different brain region, and picture is used
OLYMPUS commonly just sets microscope photographing, 5 channels, sensitivity I SO200, time for exposure 200ms.Wherein Fig. 7 A are cingulate gyrus skin
Layer cg1 and leading edge cortex Pr1；Fig. 7 B are that motor cortex M1+M2, Fig. 8 are that corpus straitum CPU, Fig. 9 A are hypothalamic area, and Fig. 9 B are
The areas incertitude zone Z1, Figure 10 A and Figure 10 B are respectively that the deadlock areas core foot interfascicular fasciculus fr and back seam nucleus DRN inversely mark result figure.It can see
Go out, viral, inflammatory reaction unobvious clear in injection site neuron morphology；Reverse projection brain area is more, projected area label
Neuron morphology it is clear, and fluorescent brightness is stronger, shows that viral reverse labeling effciency is higher.
Comparative example 1
The recombinant rabies poison that the prior art is built based on RV vaccine strain Sad B-19 infection clones, G-protein gene
It is knocked, and carries the fluorescence protein genes such as GFP, m Cherry, foreign protein high abundance in neuron can be made to express, to
The clearly fine form of labeled neurons, but be the surface glycoprotein B19G in autogene group source used in it, it is inverse
It need the space improved to the efficiency of other brain areas is marked.
RV- △ G-dsRed are virus prepared by SAD B-19.Virus is tested for live body, Figure 11 is injection site
Figure, Figure 12 to Figure 14 are the label design sketch of reverse label Different brain region, and picture is commonly just setting microscope bat with OLYMPUS
It takes the photograph, 3 channels, sensitivity I SO200, time for exposure 200ms.Wherein Figure 12 is that hypothalamus Hypothalamus, Figure 13 are outside
Hypothalamus LH, Figure 14 are that the areas back seam nucleus DRN inversely mark result figure.As can be seen that virus is in injection site labeled neurons number
Mesh is few, there is inflammatory reaction；Neuron number is less in reverse projection brain area, and projection brain area is less, shows viral reverse mark
Remember inefficient.
The present invention is replaced by targeting relevant envelope glycoprotein to virus, and virus is targeted relevant coating sugar egg
It is replaced in vain with the envelope glycoprotein N2C of another Strain, the recombination deficient mutant rabies viruses being accordingly prepared, not only
Virus yield is improved, injection site inflammatory reaction greatly reduces when live body is tested, and it inversely marks the effect of other brain areas
Rate greatly improves, and can mark full brain area neuromechanism substantially, for a kind of recombination deficient mutant of excellent label cranial nerve structure
Rabies viruses.
Sequence table
<120>A kind of rabies viruses of the full brain area neural network structure of label, it is prepared and application
<141> 2018-01-25
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2760
<212> DNA
<213> Artificial Sequence
<400> 1
gccaccatgc cagagccagc gaagtctgct cccgccccga aaaagggctc caagaaggcg 60
gtgactaagg cgcagaagaa aggcggcaag aagcgcaagc gcagccgcaa ggagagctat 120
tccatctatg tgtacaaggt tctgaagcag gtccaccctg acaccggcat ttcgtccaag 180
gccatgggca tcatgaattc gtttgtgaac gacattttcg agcgcatcgc aggtgaggct 240
tcccgcctgg cgcattacaa caagcgctcg accatcacct ccagggagat ccagacggcc 300
gtgcgcctgc tgctgcctgg ggagttggcc aagcacgccg tgtccgaggg tactaaggcc 360
atcaccaagt acaccagcgc taaggatcca ccggtcgcca ccatggtgag caagggcgag 420
gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 480
aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag 540
ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc 600
tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag 660
tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 720
tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 780
aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 840
aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc 900
aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 960
acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 1020
gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 1080
gccgccggga tcactctcgg catggacgag ctgtacaagg gaagcggagc tactaacttc 1140
agcctgctga agcaggctgg agacgtggag gagaaccctg gacctatggt tcctcaggtt 1200
cttttgtttg tactccttct gggtttttcg ttgtgtttcg ggaagttccc catttacacg 1260
ataccagacg aacttggtcc ctggagccct attgacatac accatctcag ctgtccaaat 1320
aacctggttg tggaggatga aggatgtacc aacctgtccg agttctccta catggaactc 1380
aaagtgggat acatctcagc catcaaagtg aacgggttca cttgcacagg tgttgtgaca 1440
gaggcagaga cctacaccaa ctttgttggt tatgtcacaa ccacattcaa gagaaagcat 1500
ttccgcccca ccccagacgc atgtagagcc gcgtataact ggaagatggc cggtgacccc 1560
agatatgaag agtccctaca caatccatac cccgactacc actggcttcg aactgtaaga 1620
accaccaaag agtccctcat tatcatatcc ccaagtgtga cagatttgga cccatatgac 1680
aaatcccttc actcaagggt cttccctggc ggaaagtgct caggaataac ggtgtcctct 1740
acctactgct caactaacca tgattacacc atttggatgc ccgagaatcc gagaccaagg 1800
acaccttgtg acatttttac caatagcaga gggaagagag catccaacgg gaacaagact 1860
tgcggctttg tggatgaaag aggcctgtat aagtctctaa aaggagcatg caggctcaag 1920
ttatgtggag ttcttggact tagacttatg gatggaacat gggtcgcgat gcaaacatca 1980
gatgagacca aatggtgccc tccagatcag ttggtgaatt tgcacgactt tcgctcagac 2040
gagattgagc atctcgttgt ggaggagtta gtcaagaaaa gagaggaatg tctggacgca 2100
ttagagtcca tcatgaccac caagtcagta agtttcagac gtctcagtca cctgagaaaa 2160
cttgtcccag ggtttggaaa agcatatacc atattcaaca aaaccttgat ggaggctgat 2220
gctcactaca agtcagtccg gacctggaat gagatcatcc cctcaaaagg gtgtttgaaa 2280
gttggaggaa ggtgccatcc tcatgtgaac ggggtgtttt tcaatggtat aatattaggg 2340
cctgacgacc atgtcctaat cccagagatg caatcatccc tcctccagca acatatggag 2400
ttgttggaat cttcagttat ccccctgatg caccccctgg cagacccttc tacagttttc 2460
aaagaaggtg atgaggctga ggattttgtt gaagttcacc tccccgatgt gtacaaacag 2520
atctcagggg ttgacctggg tctcccgaac tggggaaagt atgtattgat gactgcaggg 2580
gccatgattg gcctggtgtt gatattttcc ctaatgacat ggtgcagaag agccaatcga 2640
ccagaatcga aacaacgcag ttttggaggg acagggggga atgtgtcagt cacttcccaa 2700
agcggaaaag tcataccttc atgggaatca tataagagtg gaggtgagat cagactgtaa 2760
<210> 2
<211> 390
<212> DNA
<213> Artificial Sequence
<400> 2
atgccagagc cagcgaagtc tgctcccgcc ccgaaaaagg gctccaagaa ggcggtgact 60
aaggcgcaga agaaaggcgg caagaagcgc aagcgcagcc gcaaggagag ctattccatc 120
tatgtgtaca aggttctgaa gcaggtccac cctgacaccg gcatttcgtc caaggccatg 180
ggcatcatga attcgtttgt gaacgacatt ttcgagcgca tcgcaggtga ggcttcccgc 240
ctggcgcatt acaacaagcg ctcgaccatc acctccaggg agatccagac ggccgtgcgc 300
ctgctgctgc ctggggagtt ggccaagcac gccgtgtccg agggtactaa ggccatcacc 360
aagtacacca gcgctaagga tccaccggtc 390
<210> 3
<211> 717
<212> DNA
<213> Artificial Sequence
<400> 3
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717
<210> 4
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 4
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57
<210> 5
<211> 1575
<212> DNA
<213> Artificial Sequence
<400> 5
atggttcctc aggttctttt gtttgtactc cttctgggtt tttcgttgtg tttcgggaag 60
ttccccattt acacgatacc agacgaactt ggtccctgga gccctattga catacaccat 120
ctcagctgtc caaataacct ggttgtggag gatgaaggat gtaccaacct gtccgagttc 180
tcctacatgg aactcaaagt gggatacatc tcagccatca aagtgaacgg gttcacttgc 240
acaggtgttg tgacagaggc agagacctac accaactttg ttggttatgt cacaaccaca 300
ttcaagagaa agcatttccg ccccacccca gacgcatgta gagccgcgta taactggaag 360
atggccggtg accccagata tgaagagtcc ctacacaatc cataccccga ctaccactgg 420
cttcgaactg taagaaccac caaagagtcc ctcattatca tatccccaag tgtgacagat 480
ttggacccat atgacaaatc ccttcactca agggtcttcc ctggcggaaa gtgctcagga 540
ataacggtgt cctctaccta ctgctcaact aaccatgatt acaccatttg gatgcccgag 600
aatccgagac caaggacacc ttgtgacatt tttaccaata gcagagggaa gagagcatcc 660
aacgggaaca agacttgcgg ctttgtggat gaaagaggcc tgtataagtc tctaaaagga 720
gcatgcaggc tcaagttatg tggagttctt ggacttagac ttatggatgg aacatgggtc 780
gcgatgcaaa catcagatga gaccaaatgg tgccctccag atcagttggt gaatttgcac 840
gactttcgct cagacgagat tgagcatctc gttgtggagg agttagtcaa gaaaagagag 900
gaatgtctgg acgcattaga gtccatcatg accaccaagt cagtaagttt cagacgtctc 960
agtcacctga gaaaacttgt cccagggttt ggaaaagcat ataccatatt caacaaaacc 1020
ttgatggagg ctgatgctca ctacaagtca gtccggacct ggaatgagat catcccctca 1080
aaagggtgtt tgaaagttgg aggaaggtgc catcctcatg tgaacggggt gtttttcaat 1140
ggtataatat tagggcctga cgaccatgtc ctaatcccag agatgcaatc atccctcctc 1200
cagcaacata tggagttgtt ggaatcttca gttatccccc tgatgcaccc cctggcagac 1260
ccttctacag ttttcaaaga aggtgatgag gctgaggatt ttgttgaagt tcacctcccc 1320
gatgtgtaca aacagatctc aggggttgac ctgggtctcc cgaactgggg aaagtatgta 1380
ttgatgactg caggggccat gattggcctg gtgttgatat tttccctaat gacatggtgc 1440
agaagagcca atcgaccaga atcgaaacaa cgcagttttg gagggacagg ggggaatgtg 1500
tcagtcactt cccaaagcgg aaaagtcata ccttcatggg aatcatataa gagtggaggt 1560
gagatcagac tgtaa 1575
Claims (8)
1. a kind of envelope glycoprotein expression cell system, which is characterized in that it is bhk cell, and the cell line includes coating sugar egg
The virus of white gene N2C (G), envelope glycoprotein packaging can inversely absorb infection nerve cell from neuron axon end；
The cell line can express N2C (G) albumen, and it is more than generation to stablize passage 50.
2. cell line as described in claim 1, which is characterized in that the coating sugar that the envelope glycoprotein N2C is Strain CVS
Albumen.
3. cell line as described in claim 1, which is characterized in that the cell line is built to obtain by following methods：
(1) H2B-GFP-P2A-N2C (G) segment, gene order such as SEQ ID NO are obtained by PCR amplification：Shown in 1；With slow
Viral FUGW is carrier, and promoter downstream GFP genes replace with H2B-GFP-P2A-N2C (G), structure core plasmid FUGW-
H2B-GFP-P2A-N2C(G)；
(2) by the plasmid transfection 293T cells, supernatant filtering is collected in 48h~120h, obtains FUGW-H2B-GFP-P2A-
N2C (G) slow virus；
(3) by the FUGW-H2B-GFP-P2A-N2C being collected into (G) slow-virus infection bhk cell, infection multiplicity MOI is 5,48h
Observe cell state and Fluorescence Ratio after~120h and passed on, Fluorescence Ratio reaches 90% or more, pass on 5~7 times it is laggard
Row conservation obtains BHK-N2C (G) cell.
4. such as the application of claims 1 to 3 any one of them cell line, which is characterized in that be used to prepare neural markers preparation.
5. application as claimed in claim 4, which is characterized in that be used to prepare full brain area neural markers preparation.
6. a kind of rabies viruses for marking brain area nerve, which is characterized in that it is SAD-B19 attenuated strains, viral capsid
It is the envelope glycoprotein of Strain CVS containing envelope glycoprotein N2C (G), envelope glycoprotein N2C (G).
7. the preparation method of rabies viruses as claimed in claim 6, which is characterized in that include the following steps：
(1) it waits for that the passage of BHK-N2C (G) cell is stablized, 0.05% or 0.25% pancreatin of cell is digested into 2~3min, according to 1:5
~1:7 ratio is passed on, and is cultivated in 35~37 DEG C of incubators；
(2) cultivate 4-6h, abandon culture medium in culture bottle, be added RV- △ G-dsred viruses of the 13~15mL through freeze thawing and filtering into
Row infection, and be transferred in 33~35 DEG C of incubators and continue to cultivate；
(3) 48~72h is infected, when infection proportion reaches 80% or more, virocyte is subjected to digest amplification, 35~37 DEG C of cultures
Culture in case；
(4) it waits for that cell density reaches 70%-80%, cell is transferred in 33~35 DEG C of DEG C of incubators and is cultivated, and collects virus liquid
Supernatant simultaneously adds fresh culture, and virus liquid supernatant is concentrated and purified, the virus is obtained.
8. the application of rabies viruses as claimed in claim 6, which is characterized in that be used to prepare neural markers preparation, preferably use
In the label preparation for preparing the full cranial nerve of label.
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