CN101768575A - Construction of recombinant rabies virus of double-expression G gene and biological property analysis thereof - Google Patents

Abstract

The invention provides a recombinant rabies virus strain with a G gene additionally inserted in a genome, a method for constructing the recombinant rabies virus strain and application thereof. The recombinant rabies virus strain constructed by the method has high safety and immunogenicity, and is suitable for preparing vaccines for inactivating rabies virus.

Description

The structure of the recombinant rabies virus of double-expression G gene and biological property analysis thereof

Technical field:

The invention belongs to the recombiant vaccine field.Particularly, the invention provides a kind of recombinant rabies virus strain, it is the extra G gene that inserts in genome; The invention provides method that makes up described recombinant rabies virus strain and the application that is used to prepare vaccine thereof.

Background technology

Rabies are a kind of very important zoonosiss, cause 60,000 people's death every year approximately ⁽¹⁾In a lot of developing countries, dog is still causes the people to infect rabic major cause, and in developed country, rabies virus mainly is present in the wildlife host.Practice proves, and animal such as dog are carried out the sickness rate that immunity can significantly reduce human rabies ⁽²⁾, it is very important to the control rabies therefore animal to be implemented immunity.

Rabies virus belongs to the Rhabdoviridae Lyssavirus, and genome is the sub-thread strand RNA of non-segmented negative, the about 12kb of total length, the 5 kinds of viral proteins of encoding: nucleoprotein N, phosphorprotein P, stromatin M, glycoprotein G and RNA polymerase L.The glycoprotein G that is positioned on the cyst membrane is main virulence factor, also is the major antigen of inducing protective immunological reaction simultaneously.

At present, be widely used in immunity at China Flury-LEP malicious vaccine of living to dog.This virus is to equal to be separated in the lethal girl's brain of rabies in 1940 the strong malicious Flury of 1 strain by Koprowski to involve in a criminal case and continue in 1 Japanese instar chickling brain by 138 generations, passed for 40～50 generations through chick embryo yolk sac again, invade the characteristic of central nervous system when having reduced its surrounding injection.This virus is not pathogenic to dog, but keeps its immunogenicity, but still infects during the mouse intracerebral injection and cause death, and the Flury-LEP vaccine sometimes can 3 the monthly age pup cause rabies.In view of in use there is bigger potential safety hazard in attenuated live vaccines, the WHO suggestion uses rabies inactivated vaccine to replace attenuated vaccine.

For obtain efficiently, the better rabies vaccine strain of immunogenicity, this research and utilization reverse genetic technique construction a rabies virus rLEP-G who contains 2 G genes.We have identified the biological characteristics of this recombinant virus, and itself and wild-type strain Flury-LEP are compared.The result shows that recombinant virus rLEP-G compares with LEP and has better security and immunogenicity, is applicable to the exploitation of rabies virus inactivated vaccine.

Summary of the invention

At the present invention's technical problem to be solved, the present invention provides a kind of have better security and immunogenic recombinant rabies virus strain in aspect first, and it is the extra G gene that inserts in genome.

In an embodiment aspect first, the genome that comprises two G genes as shown in Figure 1, it inserts another one G gene at Pme I restriction enzyme site, inserts rabies recombinant virus genomes behind the G gene as shown in Figure 1A.In an embodiment aspect first, described recombinant virus is to be made up by wild-type rabies virus strain Flury-LEP.In an embodiment aspect first, the recombinant rabies strain of described structure is the rLEP-G that makes up as the embodiment of the invention.In one embodiment, the sequence of the G gene of described insertion is shown in SEQ ID NO:8.

In aspect second of the present invention, provide the method that makes up described recombinant rabies virus strain, described method comprises the following steps;

1) structure is transcribed plasmid, wherein the G gene order is inserted in the genome of wild-type rabies virus strain, obtains containing the recombination group full length cDNA clone of two G genes;

2) make up the helper plasmid system, described helper plasmid system encode respectively nucleoprotein N, phosphoric acid albumen P and the polymerase protein L of LEP;

3) use the plasmid vector of the recombination group full length cDNA clone contain two G genes and helper plasmid system cotransfection to the host cell of allowing described virus replication, cultivate described host cell;

4) rescue virus.

In the embodiment aspect second, the plasmid of transcribing of structure is pCI-LEP-G, and the helper plasmid of transcribing of structure is respectively pCAGG-N, pCAGG-P, pCAGG-L.In the embodiment aspect second, used host cell is the BHK-21 cell.

In aspect the 3rd of the present invention, provide the plasmid vector that comprises as the recombination group full length cDNA clone that contains two G genes constructed in the first aspect.In the embodiment aspect the 3rd, described plasmid vector is pCI.

In aspect the 4th of the present invention, be provided at the application that the recombinant virus that makes up in first aspect is used to prepare the rabies virus inactivated vaccine.

The invention effect

Quite similar by growth kinetics curve and the parent Flury-LEP strain of recombinant rabies virus strain rLEP-G strain on neurocyte NA cell and non-neurocyte BHK-21 cell that comprises two G genes provided by the invention, and the immunity test to beasle dog also shows, but the higher neutralizing antibody level of recombinant virus rLEP-G induction ratio wild-type rabies virus strain of deactivation, explanation can be used to recombinant rabies virus strain of the present invention to develop safer and have more immunogenic vaccine.

Description of drawings

The genomic structure of LEP of the two G genes of Fig. 1 (A)

(B) left figure: indirect immunofluorescence detects the viral rLEP-G of rescue and infects the BHK-21 cell,

Right figure: negative control;

Fig. 2 is wtLEP and the growth kinetics curve of rLEP-G on NA cell (A) and BHK-21 cell (B);

Fig. 3 is that rLEP-G expresses the proteic Western-blot evaluation of G

1.LEP infect the BHK-21 cell; 2.rLEP-G infect the BHK-21 cell; 3.BHK-21 cell contrast; 4. protein standard molecular weight standard

Embodiment:

The present invention will be described further by the following examples, but scope of the present invention is not limited by the examples.

Embodiment 1 contains the structure and the evaluation thereof of the recombinant virus of two G genes

1 material and method

1.1 plasmid, cell strain and virus

Plasmid pCAGG, pCI-RBz are available from Harbin Veterinary Medicine Inst., China Academy of Agriculture, and pBluescriptSK (+) is available from clonetech.BHK-21 cell (ATCC CC1-10), 293T cell (CRL-11268) are available from ATCC, and substratum is the DMEM (available from GIBCO) that contains 10% foetal calf serum.Mouse brain neuroma cell NA cell is from the military veterinary institute of Military Medical Science Institute, and substratum is the MEM (available from GIBCO) that contains 10% foetal calf serum.Rabies virus Flury-LEP and HEP increase on the BHK-21 cell available from China Veterinery Drug Inspection Office China veterinary microorganism culture presevation administrative center, and-70 ℃ frozen standby.

1.2 main agents and instrument

The T4 dna ligase, restriction enzyme Sph I, Mlu I etc. are all available from TaKaRa company.Phusion ^TMSuper fidelity dna polysaccharase.SuperScript ^TMIII reverse transcription test kit, serum free medium Opti-MEM, transfection reagent Lipofectamine ^TM2000 all available from Invitrogen company.Calf serum is available from Gibco company.The LB medium component is all available from OXOID company.Glue reclaims test kit (Gel Extraction Mini Kit) and plasmid extracts test kit (Plasmid Mini Kit) in a small amount all available from OMEGA company.Amount is extracted test kit QIAfilter Plasmid Midi Kit available from QIAGEN company in the plasmid.Mouse-anti rabies virus hyper-immune serum is by this research department's preparation.The sheep anti mouse fluorescence two of FITC mark is anti-available from Sigma company.Simple microscope is available from OLYMPUS company; Fluorescent microscope is available from ZEISS company.The PCR instrument is available from Applied Biosystem company.Nucleic acid sequencing adopts BECKMAN CEQ 8000 automatic sequencers, and agents useful for same is a BECKMAN company product.P2 level Biohazard Safety Equipment is purchased LABCONCO from company.CO2gas incubator is available from Thermo Electron company.The different model whizzer is all available from BECKMAN company.-70 ℃ of Ultralow Temperature Freezers are available from NBS company.The general refrigerator refrigerator-freezer is available from company of Haier.Automatic water purifier MILI-Q is available from BIOCEL company.All size Tissue Culture Flask and Tissue Culture Plate are all available from CORNING company.

1.3 primer

With reference to the Flury-HEP pnca gene group sequence that is provided among the Gene Bank (Gene Bank sequence number AB085828), with Flury-LEP gene order (Gene Bank sequence number DQ099524, Flury-LEP full-length gene group cDNA sequence is seen SEQ ID NO:1), with the Flury-LEP gene element be 3 sections (promptly, F1, F2 and F3) design the PCR primer respectively.Wherein, fragment F1 is the 1-4063 nucleotide sequence (SEQ ID NO:2) of Flury-LEP gene order, fragment F2 is the 4013-8254 nucleotide sequence (SEQ ID NO:3) of Flury-LEP gene order, and fragment F3 is the 8187-11925 nucleotide sequence (SEQ ID NO:4) of Flury-LEP gene order.Make up full-length gene group cDNA the primer, introduce Pme I the primer (table 1) and make up helper plasmid the primer (table 2) synthetic by the handsome Bioisystech Co., Ltd in Shanghai.

Table 1 RT-PCR and full-length gene group cDNA make up used primer

Annotate: the cDNA segment is consistent with Fig. 1.Underscore is viral distinguished sequence partly, and restriction enzyme site represented in black matrix, and hammerhead ribozyme (HamRz) and fourth hepatovirus ribozyme (HdvRz) are represented with italic.

Table 2 helper plasmid makes up used primer

Annotate: underscore is viral distinguished sequence partly, and restriction enzyme site represented in black matrix, and mutating alkali yl represents that with the character frame Kozak sequence is represented with italic.

1.4 make up Flury-LEP genome full length cDNA clone and cDNA fragment cloning and helper plasmid system

1.4.1 virus genomic extraction, reverse transcription and PCR

Get Flury-LEP virus liquid 250 μ L, add 750 μ L Trizol, extract geneome RNA through conventional method.The RNA SuperScript that extracts ^TMIII reverse transcription test kit carries out reverse transcription reaction (the reverse transcription primer sees Table 1) to be operated by this test kit specification sheets, obtains terminal portions eclipsed 3 viral cDNA fragment F1, F2 and F3 fragment.

1.4.2 the clone of RT-PCR product

Whole genome is divided into 3 segments of terminal portions eclipsed and carries out the RT-PCR amplification, the PCR product is through 1% agarose gel electrophoresis, use glue to reclaim test kit (available from OMEGA company) and reclaim F1, F2 and F3 fragment, be cloned into pBluscript II KS (+/-) EcoR v site (Clontech) respectively, transformed competence colibacillus cell DH5 α, select and extract positive plasmid pBlue-F1, pBlue-F2 and pBlue-F3, cloned genes group fragment and virus genome RNA sequence are guaranteed in order-checking, and in full accord (concrete grammar is seen " molecular cloning test guide ", J. Sa nurse Brooker is compiled, the third edition in 2008, Science Press translates).

1.4.3 the structure of Flury-LEP genome full length cDNA clone and cDNA fragment cloning

The restriction site that utilizes the adjacent lap with F3 of genome cDNA fragment F1, F2 to exist assembles (synoptic diagram is seen Fig. 1).Particularly, at first F3 section cDNA is cloned into pCI-RBz through Nhe I and Rsr II site, gained plasmid called after pCI-F3 is inserted into pCI-F3 with the F1 section through Nhe I and Mlu I again, gained plasmid called after pCI-F31.At last the F2 section is cloned into pCI-F31 through Sph I and Mlu I, the full genome cDNA assembling of Flury-LEP is so far finished, and the viral genome that is built into is transcribed plasmid called after pCI-LEP.

This is cloned under the CMV promotor, the full-length cDNA between two ribozymes can be transcribed under the effect of eukaryotic cell RNA polymerase, and because the autocatalysis function of ribozyme can guarantee that 3 of transcription product ' end is accurately consistent with viral genome.

1.4.4 contain the structure of the genome full-length cDNA of two G genes

Obtain the reorganization LEP virus of expressing two G genes in order to rescue, transcribe in viral genome on the basis of plasmid pCI-LEP, at first be structured in the full length cDNA clone that has Pme I restriction enzyme site between G and the L gene, concrete grammar is: be template with pCI-LEP, utilize F2-PF and Pme I-PR to be primer, amplifying PCR fragment PmeA, is that primer amplification goes out PCR fragment PmeB with F2-PR and Pme I-PF; And then be template with PCR fragment PmeA and PmeB, be primer with F2-PF and F2-PR, amplify and contain the F2 fragment that Pme I enzyme is cut, called after F2-P.F2-P is cloned into pCI-F31 through SphI and Mlu I, thereby be built into recombination group transcription vector pCI-LEP-PmeI.Then, with upstream primer I nsertGPF:5 '-ATGC GTTTAAACAAGTTTATCACTTGTTTACCTCT-3 ', downstream primer InsertGPR:5 '-GCAT GTTTAAACACTTGAAGTGTCAAAAGGATGA-3 ' (underscore is a restriction enzyme site Pme I sequence) is that template amplification goes out the G gene fragment 1800bp (SEQ IDNO:8) that two ends have Pme I restriction enzyme site, include transcriptional initiation sequence, transcription termination sequence with pCI-LEP-Pme I.Through check order errorless after, this PCR product end is cut through Pme I enzyme and is inserted equally in the pCI-LEP-Pme I carrier that Pme I handles the recombination group full length cDNA clone called after pCI-LEP-G that contains two G genes that builds.

1.4.5 make up the helper plasmid system that transcribes

With pCI-LEP is template, (nucleotide sequence is respectively SEQ ID NOs:5-7 to open reading frame (ORF) the cDNA sequence of pcr amplification coding nucleoprotein (N), phosphoric acid albumen (P) and polymerase protein (L) gene, primer sequence sees Table 2), be cloned in the multiple clone site (MCS) of pCAGG plasmid respectively.Before initiator codon, introduce Kozak sequence (GCCACC), to strengthen protein expression; After original terminator codon, increase a terminator codon, with transcribing of effective termination helper plasmid mRNA.The helper plasmid that is built into is called after pCAGG-N, pCAGG-P and pCAGG-L respectively.

1.5 virus rescue

Transfection the day before yesterday, the BHK-21 cell is passed on 35mm 6 orifice plates, when reaching 80%, cell density can carry out transfection when above.PCI-LEP-G (seeing 1.3), pCAGG-N, pCAGG-P, pCAGG-L are joined among the 250 μ l serum free medium OPTI-MEM (available from GIBCO company) mixing with the ratio of 4 μ g, 2 μ g, 1 μ g, 1 μ g respectively; Get 15 μ l Lipofectamine ^TM2000 are dissolved among the 250ul OPTI-MEM, leave standstill 5min in room temperature behind the mixing; Then with Lipofectamine ^TM2000 mix incubated at room 30min with transfection plasmid (being pCI-LEP-G, pCAGG-N, pCAGG-P, pCAGG-L each 4 μ g, 2 μ g, 1 μ g, 1 μ g).With OPTI-MEM washing BHK-21 cell twice, every then hole adds OPTI-MEM 1ml, at last with Lipofectamine during this period ^TM2000-plasmid mixed solution adds in the cell cultures hole, places 37 ℃ of CO ₂Transfection liquid is abandoned in suction after cultivating 8h in the incubator, adds the DMEM that contains 10% foetal calf serum and continues to cultivate.After 72 hours, the cell on the culture plate that carefully blows off is all inoculated the BHK-21 cell with nutrient solution and cell mixture, cultivates 3 days for 37 ℃.After 3 days, cell conditioned medium is done 10 ^-1-10 ^-5The inoculation of dilution back grows in the BHK-21 cell of 24 orifice plates, after 2 days, detect virus with indirect immunofluorescence assay and whether save success, be one anti-promptly with the mouse-anti rabies virus hyper-immune serum of dilution in 1: 50, mouse negative serum with the dilution of identical multiple is contrast, is two anti-with the sheep anti-mouse igg (Sigma) of fluorescein (FITC) mark.According to the titre of rescue virus, carry out the suitable proportion diluted passage, preparation kind of poison.Rescuing the viral nomenclature that obtains from total length plasmid pCI-LEP-G is rLEP-G.

1.6 RT-PCR identifies and sequencing

Recombinant virus is carried out sequential analysis: get recombinant virus 250 μ l, extract viral RNA, use upstream primer with viral RNA (in a small amount) extraction agent box

5 '-CAATCCTTGGGAGCAATAGA-3 ' and downstream primer

5 '-CGTTGGTCACTGAAACTGCTA-3 ', carry out the fragment that the amplification of RT-PCR method contains two G genes, and amplification PCR products is carried out sequential analysis.

1.7 plant the preparation and the titration of poison

The recombinant virus of above-mentioned rescue is increased on the BHK-21 cell, cultivated 3 days for 37 ℃.Virus titer is measured in results virus back, is about to virus and is diluted to 10 for continuous 10 times with aseptic PBS ^-8, each extent of dilution 3 multiple hole inoculation culture is in the individual layer NA cell or the BHK-21 cell of 24 orifice plates.After cultivating 48h, carry out IFA, virus titer is represented with the FFU of the focus unit of formation (focus forming unit).

1.8 the mensuration of multistep growth curve

For measuring the multistep growth curve of virus, be 0.01 infection individual layer NA cell or BHK-21 cell with M.O.I respectively.Usefulness behind the 1h is made in sense, and serum-free MEM (available from GIBCO) washes the MEM that adds 0.2%FBS after 2 times or 2%DMEM in 37 ℃ of cultivations.Get the cell culture fluid supernatant respectively at inoculation back 24 hours, 72 hours and 120h, on NA cell or BHK-21 cell, measure titre.

1.9 recombinant virus is to the pathogenic test of mouse

Make 10 times of doubling dilutions to 10 with rescuing the virus and the wild virus L E P that obtain with sterilization PBS ^-7, with 10 ^-4～10 ^-7Each extent of dilution is got the Balb/c female mice in age 5 5～6 weeks of 30 μ l intracranial inoculations respectively.Observed 21 days continuously, the mean body weight that writes down every group of mouse changes and dead quantity.Utilize the Reed-Muench method to calculate mouse medium lethal dose (LD ₅₀) ⁽⁵⁾

With 10 ⁷The rLEP of FFU and rLEP-G be the Balb/c female mice in age 4 5～6 weeks of mouse back leg intramuscular injection respectively, observes continuously 21 days, and the mean body weight that writes down every group of mouse changes and dead quantity.

1.10 Western-blot detects recombinant virus G genetic expression

Recombinant virus rLEP-G, wild-type LEP are 1 to infect the BHK-21 cell with M.O.I, and collecting cell carries out the SDS-PAGE electrophoresis after 3 days, with blank cell sample in contrast.To the NC film, 10% skim-milk sealing is spent the night with the albumen electrotransfer, is one anti-with the mouse-anti G albumen hyper-immune serum of dilution in 1: 50, the IRDye of dilution in 1: 5000 ^TMThe sheep anti-mouse igg of 700DX mark is two anti-.Carry out interpretation of result with Odyssey Infrared fluorescence scanning imaging system.

2. result

2.1 contain LEP full-length gene group cDNA clone's the structure of two G genes and the rescue of recombinant virus

Through PCR method, amplify the G gene fragment that two ends have Pme I restriction enzyme site, include transcriptional initiation sequence, transcription termination sequence, this PCR product end is cut through Pme I enzyme and is inserted equally on the pCI-LEP-Pme I that Pme I handles, and is built into the reorganization LEP cDNA cloned plasmids pCI-LEP-G (Fig. 1) that contains two G genes.

In order to save infectious recombinant virus from the cDNA clone, at first with pCI-LEP-G and express RVN, P, the proteic helper plasmid cotransfection of L BHK-21 cell, harvested cell and supernatant liquor after 3 days, IFA tests positive result (Figure 1A) after the blind passage generation, and virus titer reaches 2 * 10 ⁴FFU/ml, preparation kind of poison continues to go down to posterity on the BHK-21 cell.For whether the virus of determining rescue derives from pCI-LEP-G, recombinant virus is carried out RT-PCR identify that amplify the 3732bp fragment, this fragment includes 2 G gene orders.Further order-checking proof, the virus that obtains of rescuing is from pCI-LEP-G.

2.2 the biological characteristics of recombinant virus

2.2.1 kinetic curve

RLEP-G and the LEP growth kinetics curve on neuronal cell NA and non-neuronal cell BHK-21 is (Fig. 2) as shown in the figure, growth performance and the wtLEP of rLEP-G on two kinds of cells do not have significant difference, and these growth characteristics that show the rLEP-G that saves out from the genome full-length cDNA are consistent with wtLEP.

2.2.2 have a liking for neural index

Rabies virus external have a liking for neural index be meant virus at the NA cell with respect to the infectious ratio on the BHK-21 cell, have a liking for neural index and be 1 i.e. expression virus the energy force rate that infects on the neuronal cell high 10 times on non-neurocyte ^[6]The neural index of having a liking for of visible rLEP-G of table 1 and wtLEP is respectively 0.85 and 0.84, does not have significant difference.

Table 1 wtLEP and LEP-G external had a liking for nervosa

A is with the titre of a kind of poison on NA cell and BHK-21 cell

B is external to have a liking for neural index=log (titre on the NA)-log (titre on the BHK-21)

2.2.3 pathogenic to mouse

In order to determine pathogenic to mouse of rLEP-G and wild-type LEP, virus inoculation 10 in every mouse brain ⁵FFU/30 μ l.The clinical symptom of central nervous system diseases such as the mouse that infects rLEP-G and LEP shows paralysis, excitement, lose weight, all mouse are whole death in 11 days.In addition, we have measured the LD of wtLEP and rLEP-G ₅₀, be respectively 8.8 FFU and 3.4 FFU.As seen, rLEP-G and wtLEP do not have significant difference to mouse pathogenic.But, 10 ⁷The LEP periphery infecting mouse of FFU can cause mouse invasion and cause death, as seen the also residual certain nerve of LEP is encroached on ability, and the identical titre periphery of rLEP-G strain infecting mouse causes mouse weight loss and indivedual back acroparalysia but does not cause dead mouse, and the security that shows rLEP-G is than the LEP height.

2.2.4Western-blot detect recombinant virus G genetic expression

Western-blot analyzes rLEP-G and infects the expression level of BHK-21 cell in cell.RLEP-G, wtLEP is 1 infection BHK-21 cell collecting cell cracking after 3 days with M.O.I, as Fig. 3, is confidential reference items with β-action, the G expression amount of rLEP-G is significantly higher than the G expression amount of wtLEP.

Embodiment 2 recombinant viruses are to the immunity test of beasle dog

In order to estimate the immune effect of recombinant virus, with 8 * 10 to dog ⁷The wtLEP of FFU/ml and rLEP-G use 1: 6000 β-third lactones to handle the 2h deactivation respectively, behind 3: 1 mixings of aluminum phosphate adjuvant (available from REHEIS company), through each 6 of intramuscular injection path immunity 3 monthly age beasle dogs (available from Test Animal Centre, Academy of Military Medical Sciences, P.L.A), establish 6 simultaneously and be contrast, intramuscular injection 1ml PBS.Immunity blood sampling in back 21 days and separation of serum are used for NAT and detect.Neutralization test

At first 56 ℃ of water bath processing 30min deactivation of serum sample, continuous subsequently 20 times to 1280 times doubling dilutions are established the contrast of positive and negative serum simultaneously.Virus adopts standard fixed poison CVS strain, and the serum that dilution is good mixes with the viral equal-volume that contains 1000 FFU, hatches 1h for 37 ℃, gets then on the 24 hole BHK-21 cells that 100 μ l are added to overnight growth.Each sample is done 3 repetitions.Infect back 48h and carry out the IFA detection.The NAT of tested serum is to make the fluorescence kitchen range suppress to equal 50% the highest multiple of serum.The initial dilution of positive control serum (available from rabies OIE reference laboratory, France) is tired and is 2IU/ml, in antibody unit to be measured (IU/ml)=test serum and in titre/standard serum and titre * 2.

Recombinant virus rLEP-G is to the immune protective of beasle dog

Recombinant virus rLEP-G and wild-type wtLEP according to O.I.E standard test deactivation induce the horizontal experimental result of beasle dog neutralizing antibody to show: deactivation rLEP-G immunity beasle dog induces the neutralizing antibody of generation to reach 14 ± 4IU/ml, it is 6 ± 2.3IU/ml that deactivation wtLEP immunity beasle dog is induced the NAT of generation, two groups of data differences remarkable (P＜0.05).As seen, but the higher neutralizing antibody level of recombinant virus rLEP-G induction ratio wild-type of deactivation more is applicable to the exploitation of rabies virus inactivated vaccine.

Discuss

This research is successfully saved recombinant rabies virus rLEP-G strain by the reverse genetic technology, this recombinant virus is on parent's attenuated vaccine strain Flury-LEP strain reverse genetic operating system basis, and the G gene that make up to strengthen a virus itself promptly contains the viral full length cDNA clone of 2 identical G genes and rescue acquisition.The Flury-LEP strain has good immunogenicity, is widely used as human or animal's rabies inactivated vaccine kind strain.Also comprised the attenuated vaccine of many countries of China as the prevention rabies.

Growth kinetics curve and the parent Flury-LEP strain quite similar (Fig. 2) of rLEP-G strain on neurocyte NA cell and non-neurocyte BHK-21 cell, however rLEP-G proteic expression amount of G in the BHK-21 cell is significantly higher than parent plant LEP (Fig. 3).

Our experimental result has proved that also G gene of extra increase does not influence the growth performance of virus, and the proteic expression amount of G significantly improves in cells infected.Immunity test to beasle dog also shows, but the higher neutralizing antibody level of recombinant virus rLEP-G induction ratio wild-type of deactivation more is applicable to the exploitation of rabies virus inactivated vaccine.

Membrane glycoprotein G albumen is the topmost virulence factor of rabies virus ^(6,7)Still infect during the intracranial inoculation of LEP strain mouse and cause death, additionally increase a G albumen and whether can strengthen its pathogenecity.By measuring LD50 and observing the mouse state, we find the LD of rLEP-G strain ₅₀LEP does not have significant difference with its parent plant, and this shows that the virulence of rLEP-G strain almost is the same with parent plant LEP.The high titre periphery of LEP infecting mouse can cause mouse invasion and cause death, the also residual certain nerve infringement of visible LEP ability, and rLEP-G plant height titre periphery infecting mouse causes mouse weight loss and indivedual back acroparalysia but does not cause dead mouse.Influencing one of them pathogenic factor of RV is to escape apoptotic ability ⁽⁸⁾The recombinant virus cell death inducing ability that increases the G protein content strengthens, virulence to mouse when periphery infects descends, correspondingly be that the NAT of virus significantly rises, this may be because maturation and the antigen presentation that the release of endogenous adjuvant has stimulated the cytotoxic T cell reaction and can promote dendritic cell.

The rna gene group of rabies virus exists with the RNP composite form, and rna gene group template and transcripton thereof are all closely wrapped up by N albumen, makes it not be subjected to the degraded of RNA enzyme, keeps high reactivity.Mebatsion etc. ⁽⁹⁾The recombinant virus of Zheng Jiu expression foreign protein CAT goes down to posterity and still can detect the CAT activity after 25 times first.The high genetic stability of the reorganization RV rna gene group after the insertion external source fragment is one of its advantage of using as carrier.The rLEP-G strain is still possessed extra G gene and is kept the high titre and the G albumen high expression level of virus after continuous several times goes down to posterity on the BHK-21 cell, show the genetic stability of rLEP-G strain in culturing cell.

Reference

1.Martinez?L.Global?infectious?disease?surveillance.Int?J?Infect?Dis，2000，4(4)：222-8.

2.Kammer?AR，Ertl?HC.Rabies?vaccines：from?the?past?to?the?21st?century.Hybrid?Hybridomics，2002，21(2)：123-7.

3.Faber?M，Pulmanausahakul?R，Hodawadekar?SS，et?al.Overexpression?ofthe?rabies?virus?glycoprotein?results?in?enhancement?of?apoptosis?andantiviralimmune?response.J?Virol，2002，76(7)：3374-81.

4.Hosokawa-Muto?J，Ito?N，Yamada?K，et?al.Characterization?of?recombinantrabies?virus?carrying?double?glycoprotein?genes.MicrobiolImmunol，2006，50(3)：187-96.

5.Reed，LJ，Muench，H.A?simple?method?of?estimating?fifty?percent?endpoints.Am.J.Hyg，1938，27：493-497.

6.Morimoto?K，Hooper?DC，Carbaugh?H，et?al.Rabies?virus?quasispecies：implications?for?pathogenesis.Proc?Natl?Acad?Sci?USA，1998，95(6)：3152-6.

7.Dietzschold?B，Wunner?WH，Wiktor?TJ，et?al.Characterization?of?anantigenic?determinant?of?the?glycoprotein?that?correlates?with?pathogenicityof?rabies?virus.Proc?Naal?Acad?Sci?USA，1983，80(1)：70-4.

8.Morimoto?K，Hooper?DC，Spitsin?S，et?al.Pathogenicity?of?different?rabiesvirus?variants?inversely?correlates?with?apoptosis?and?rabies?virusglycoprotein?expression?in?infected?primary?neuron?cultures.J?Virol，1999，73(1)：510-8.

9.Mebatsion?T，Schnell?MJ，Cox?JH，et?al.Highly?stable?expression?of?aforeign?gene?from?rabies?virus?vectors.Proc?Natl?Acad?Sci?USA，1996，93(14)：7310-4.

Sequence table

＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture

＜120〉structure of the recombinant rabies virus of double-expression G gene and biological property analysis thereof

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acgaaaagga?gacaagatca?ccccagactc?tctagtggag?ataaagcgta?ctgatgtaga 420

agggaattgg?gctctgacag?gaggcatgga?actgacaagg?gaccccactg?tctccgaaca 480

tgcatcttta?gtcggtcttc?tcctgagtct?gtacagattg?agcaaaatat?caggacaaaa 540

cactggtaac?tataagacaa?acattgcgga?tagaatagag?cagattttcg?agacagcccc 600

ttttgttaag?atcgtggaac?accataccct?gatgacaact?cacaagatgt?gtgctaattg 660

gagtactata?ccgaacttca?gatttttggc?cggaacctac?gacatgtttt?tctcacggat 720

cgagcatctg?tattcggcaa?tcagagtggg?cacagttgtc?accgcttatg?aagactgctc 780

aggactggta?tcgtttacag?ggttcataaa?gcagatcaat?ctcaccgcaa?gagaagcaat 840

actatatttc?ttccacaaga?actttgaaga?agagataagg?agaatgtttg?agccagggca 900

agagacagct?gttcctcact?cttatttcat?tcacttccgt?tcactaggct?tgagtgggaa 960

gtctccttat?tcatcaaatg?ccgtcggtca?tgtgttcaat?ctcattcact?ttgttggatg 1020

ctatatgggt?caagtcagat?ctctaaatgc?aacggttatt?gctgcatgtg?cccctcatga 1080

gatgtctgtt?ctagggggct?atttgggaga?ggagttcttc?ggaaaaggga?catttgaaag 1140

aaggttcttc?agagacgaga?aagaacttca?agaatatgag?gcggctgaac?tgacaaagac 1200

cgacgtggca?ctggcagatg?acggaaccgt?caactccgat?gacgaggact?acttctccgg 1260

tgaaaccaga?agtccagaag?ctgtctatac?tcgaatcatg?atgaatggag?gtcgactgaa 1320

gagatcacat?atacggagat?atgtctcagt?cagttccaat?catcaagccc?gtccaaactc 1380

attcgccgaa?tttttaaaca?aaacgtattc?gagtgactca?taaggagttg?aatgacaggg 1440

tgccagaaat?ccatagattg?tgtatatcca?tcatgaaaaa?aactaacact?cctcctttcg 1500

aaccatccca?agtatgagca?agatctttgt?taatccgagt?gcaatcagag?ccggtctggc 1560

cgatcttgag?atggccgaag?agactgttga?tctgatcaac?agaaacatag?aagacaatca 1620

ggctcatctc?cagggagaac?ccatagaagt?ggacaatctc?cctgaggaca?tgaggcaatt 1680

tcacctgggc?gatgaaaaat?tgtccaacct?tggtgagatg?gttagggtgg?gcgaaggcaa 1740

gtatcgagag?gactttcaga?tggatgaggg?agaggacccc?aacctcctgt?tccaatcgta 1800

cctggacaat?gttggagtcc?aaatagtcag?acaaatgagg?tcaggagaga?gattcctcaa 1860

gatatggtca?cagaccgtag?aggaaattat?atcctatgtc?acggtcaact?ttcctaaccc 1920

tccaggaagg?tcttcggagg?ataaatcaac?ccaaactact?ggccgggagc?tcaagaagga 1980

gacaacatcc?actctttctc?agagagaaag?ccaaccttca?aaagccggaa?tggtggctca 2040

agttgcctct?ggccctccat?cccttgaatg?gtctgccacc?aatgaagagg?atgatctatc 2100

agtagaggct?gagatcgctc?atcagattgc?tgaaagcttt?tccaagaagt?acaagtttcc 2160

ctctcgatct?tcaggaatat?tcttgtataa?ttttgagcaa?ctggagatga?accttgatga 2220

catagttaaa?gaggcaaaaa?atgtaccggg?cgtgacccgt?ctggcccatg?atggatccaa 2280

aatcccccta?agatgtgtac?tgggatgggt?cgctttggcc?aactccaaga?aattccaatt 2340

gatagtcgag?gccgacaagc?taagcaaaat?catgcaagat?gacttggatc?gctacacatc 2400

atgctaaccg?agtcttcgaa?ttcattccct?ctagataatg?aaaactgaga?tgtcatggag 2460

tcgacatgaa?aaaaacaggc?aacaccacta?ataaaatgaa?ctttctatgt?aagatagtga 2520

aaaactgtag?ggatgaggac?acccaaaagc?cctctcctgt?gtcagcccct?ccggatggcg 2580

atgacctgtg?gcttccacct?ccagaatatg?tcccgctgaa?agaactcaca?agcaagaaga 2640

acatgaggaa?cttttgtatc?aacggggagg?ttaaagtgtg?tagcccgaac?ggttactcat 2700

tcaggatcct?gcggcatatt?ctgagatcat?tcgacgagat?atactctggg?aatcatagga 2760

tgattgggtt?agtcaaagtt?gttattggac?tagctttatc?aggagctcca?gttcctgagg 2820

gcatgaactg?ggtatacaaa?ttgaggagaa?cccttatttt?ccagtgggct?gattccaggg 2880

gccctcttga?aggggaggag?ttggaacact?ctcaagagat?cacttgggac?gatgatactg 2940

aattcgtcgg?attgcaaatg?agagtgagcg?caagacaatg?tcatattcaa?ggcaggatct 3000

ggtgtatcaa?catgaactcg?agggcatgtc?aactatggtc?tgacatgtct?cttcagacac 3060

aaaggtctga?agaggacaaa?gactcttctc?tgcttctaga?ataatcagat?tatatcccgc 3120

aagtttatca?cttgtttacc?tctggaggag?agaacatacg?ggcttaactc?caatccttgg 3180

gagcaataga?acaaaaaaac?acgccatggt?gccattaaac?cgctgcattt?tatcaaagtc 3240

aagttaatta?cctttacatt?ttgagcctct?tggatgtgaa?aaaaactatt?aacatccctc 3300

aaaagactta?aggaaagatg?gttcctcagg?ttcttttgtt?tgtacccctc?ctgggttttt 3360

cattgtgttt?cgggaagttc?cccatttaca?cgataccaga?caaacttggt?ccctggagcc 3420

ctattgacat?acaccatctc?agctgtccaa?ataacctggt?tgtggaggac?gaaggatgta 3480

ccaacctgtc?cgagttctct?tacatggaac?ttaaagtggg?atacatctca?gccataaaag 3540

tgaacgggtt?cacttgcaca?ggtgttgtga?cagaggcaga?aacctacacc?aactttgttg 3600

gttatgtcac?aaccacattc?aagagaaagc?atttccgccc?caccccagac?gcatgtagag 3660

ccgcgtataa?ctggaagatg?gccggtgacc?ccagatatga?agagtctcta?cacaatccgt 3720

accccgacta?ccattggctt?cgaactgtaa?aaaccaccaa?agagtctctc?gttatcatat 3780

ccccaagtgt?gacagatttg?gacccatatg?acaaatccct?tcactcaagg?gtcttccctg 3840

gcggaaattg?ctcaggaata?acggtgtcct?cgacctactg?ctcaactaat?catgattaca 3900

ccatttggat?gcctgagaat?ctgagactag?ggacatcttg?tgacattttt?accaatagca 3960

gagggaagag?ggcatccaaa?ggaggcaaga?cttgcggctt?tgtggatgaa?agaggcctgt 4020

ataagtctct?aaagggagca?tgcaaactca?agttatgtgg?agttctcgga?cttagactta 4080

tggatggaac?atgggtcgcg?atgcaaacat?cagatgagac?caaatggtgc?cctccaggtc 4140

agttggtgaa?tttgcacgac?tttcgctcag?acgagattga?gcatctcgtt?gtggaagagt 4200

tagtcaagaa?aagagaggag?tgtctggatg?cactagagtc?catcatgacc?accaagtcag 4260

tgagtttcag?acgtctcagt?cacttgagaa?aacttgtccc?tgggtttgga?aaagcatata 4320

ccatattcaa?caaaaccttg?atggaggctg?atgctcacta?caagtctgtc?cggacctgga 4380

atgagatcat?cccctcaaaa?gggtgtttga?gagttggggg?gaggtgtcat?ccccatgtga 4440

acggggtgtt?tttcaatggt?ataatattag?ggtctgacgg?ccatgttcta?atcccagaga 4500

tgcagtcatc?cctcctccag?caacatatgg?agttgttgga?atcttcagtt?atccccctga 4560

tgcacccctt?ggcagaccct?tctacagttt?tcaaagacgg?tgatgaggtt?gaggattttg 4620

ttgaagttca?cctccccgat?gtgcatgaac?aggtctcagg?agttgaactg?ggtctcccga 4680

actgggggaa?gtatgtattg?atgattgcag?gggccttgat?tgccctgatg?ttgataattt 4740

tcctgatgac?atgttgcaga?agagtcaatc?gaccagaatc?tacgcaaagc?agtcttggag 4800

agacagggag?aaatgtgtca?gtcacttccc?aaagcggaaa?agtcatatct?tcatgggagt 4860

catataagag?tggaggcgag?accagactgt?gaaggccggt?catccttttg?acacttcaag 4920

tcccgaggat?aacctcctct?cggggttggg?gggaatcttg?ggatccagta?gtcctccttg 4980

aactccatcc?aacagggtag?atttaagagt?catgagactt?tcattaatca?tctcagttga 5040

tcagacatgg?tcgtgtagat?tctcataaca?cgggagatct?tctagcagtt?tcagtgacca 5100

acggtgcttt?cattctccag?gaactgatac?caaaggttgt?ggacaagcca?aggggtgctt 5160

cggattactc?tgtgcttggg?cacagaaaga?ggtcgtagtt?tgccccttga?tagcagattc 5220

aacatgaatt?aactaagaaa?ggcgatctgc?ctcccatgaa?ggacataagc?aatagttcac 5280

aatcatcttg?catctcagtg?aagtgtacat?aactataaag?ggctgggtca?tctaagcatt 5340

tcagtcgaga?aaaaaactgt?agaccaaaag?aacaactaac?aacacttctc?atcccgagac 5400

ccatatcaag?atgctggatc?cgggagaggt?ttatgatgac?cctattgatc?caattgagtc 5460

agaggctgaa?cccagaggaa?cccccactgt?ccccaacatc?ttgaggaact?ctgactacaa 5520

tctcaactct?cctttgatag?aggactctgc?caaactaatg?ttagaatggt?tgaaaacagg 5580

gaacagacct?tatcggatga?ctttgacaga?caattgctcc?aggtcttaca?aagttttgaa 5640

agattatttc?aagaaagtag?atttgggttc?tctcaaagtg?ggaggaactg?ctgcacaatc 5700

aatgatttct?ctctggttgt?atggagccca?ctctgagtca?aacaggagcc?ggagatgtat 5760

aaccgacttg?gcccatttct?attccaagtc?atcccccata?gagaagctgt?tgaattgtac 5820

gttaggaaac?agaggcctga?gaatcccacc?agagggggtg?ttaagttgcc?ttgagagggt 5880

cgattatgat?aaggcatttg?ggaggtatct?ggccaacacg?tattcctctt?atctgttttt 5940

ccatgtaatt?accttataca?tgaatgccct?agactgggaa?gaggaaaaaa?ccatcctagc 6000

attatggaaa?gatctaacct?cagtggatac?cgggaaggac?ttggtcaaat?tcaaagatca 6060

aatatgggga?ctgctggttg?tgacaaagga?ctttgtttac?tctcagagtt?ctaactgtct 6120

ttttgacaga?aactatacac?tgatgctaaa?ggatcttttc?ttgtctcgat?tcaactcctt 6180

gatgattttg?ctttctcccc?ctgagccccg?atactcagat?gacttaatat?ctcagctgtg 6240

ccagctatac?atcgctgggg?atcaagtctt?gtccttgtgt?gggaactccg?gctatgaagt 6300

catcaaaata?ttggagccat?atgtcgtgaa?cagtttggtc?cagagggcag?agaagtttag 6360

gcctctcatc?cactccttgg?gagactttcc?tatgtttata?aaagacaagg?tgaatcaact 6420

tgaagggact?ttcggtccca?gtgcaaaaag?gttttttagg?gttctagatc?aattcgacaa 6480

catacatgat?ctagtatttg?tgtatggctg?ttacagacat?tgggggcacc?cctatataga 6540

ttatcggaag?ggtctgtcga?aactatatga?tcaagttcac?attaagaaag?taatagataa 6600

gtcctaccag?gagtgtttag?caagtgactt?ggctagaagg?atcctcagat?ggggatttga 6660

caagtactcc?aagtggtatc?tagattcgag?attccttgcc?ctagaccacc?ccttggctcc 6720

ttatatcaag?acccaaacat?ggccacccaa?acatatagta?gacttggtgg?gggacacatg 6780

gcacaagctc?ccgatcacgc?agatctttga?gattcctgaa?tcaatggacc?cgtcagagat 6840

actggatgat?aaatcacatt?ctttcaccag?aacaagacta?gcttcttggc?tgtccgagaa 6900

ccgagggggg?cctgttccta?gcgagaaggt?cattatcacg?gccctgtcta?agccacctgt 6960

caatccccga?gagtttttga?aatctatcga?cctcggagga?ttgccagatg?atgacttgat 7020

aattggcctc?agaccaaagg?aacgggagtt?gaagattgag?ggccgattct?tcgctctaat 7080

gtcatggaat?ctaagattat?attttgtcat?caccgagaag?ctcttggcca?actacatttt 7140

gccacttttt?gacgcactga?ctatgacaga?caacctgaac?aaggtattta?aaaagttgat 7200

cgacagggtc?accgggcaag?ggcttttgga?ctattcgagg?gtcacatacg?catttcacct 7260

ggactatgag?aaatggaaca?atcatcaaag?attggagtca?acggaggatg?tattttctgt 7320

cctagatcag?gtgtttggat?tgaagagggt?gttttctaga?acacacgagt?tttttcagaa 7380

gtcctggatc?tattattcag?acagatcaga?cctcattggg?ttacgggagg?atcagatata 7440

ttgcttggat?atgtccaatg?gtccaacctg?ctggaatggc?caagacggcg?ggctagaggg 7500

cttacggcag?aagggctgga?gtctagtcag?cttattgatg?atagatagag?aatctcaaac 7560

caggaacaca?agaaccaaga?tactagctca?aggagacaac?caggttctgt?gtccgacata 7620

catgttgtca?ccgggattgt?ctcaagaggg?gcttctctat?gagttagaga?gcatatcgag 7680

gaatgcactc?tcaatatacc?gagctatcga?ggaaggagca?tctaaactgg?ggctgatcat 7740

caagaaagaa?gagaccatgt?gtagttatga?ctttctcata?tatgggaaga?cccccttatt 7800

tcgaggcaac?atattagtac?ctgaatccaa?aagatgggcc?cgagtctctt?gcatctctaa 7860

cgaccaaata?gtcaacctcg?ccaatataat?gtctacagta?tccaccaatg?cgctgacagt 7920

ggcacaacac?tctcaatctc?tgatcaaacc?tatgagggat?tttctgctca?tgtcagtaca 7980

ggcagttttc?cactacctgc?tgtttagccc?aatcttaaaa?ggcagggttt?ataagattct 8040

gagtgctgaa?ggggagagct?ttctcctagc?catgtcgcgg?ataatctacc?tagatccttc 8100

tttgggaggg?gtgtctggaa?tgtctctcgg?gaggttccat?atacgtcagt?tctcagaccc 8160

tgtctctgaa?gggttgtcat?tctggagaga?gatctggtta?agctcccatg?aatcctggat 8220

tcacgcgttg?tgtcaagagg?ccgggaaccc?cgatcttgga?gagagaacac?tagagagctt 8280

cactcgcctt?ttagaagatc?ctactacctt?aaatatcaaa?ggaggggcca?gtcctaccat 8340

tctactcaag?gatgctatca?gaaaggctct?gtacgacgag?gtggacaagg?tggaaaattc 8400

agagtttcga?gaggcaatcc?tgttgtccaa?gacccataga?gataacttta?tactcttttt 8460

aaaatctgtt?gagcctctgt?tccctcgatt?tctcagtgaa?ctcttcagtt?cgtccttctt 8520

gggaatacca?gagtcaatta?ttggactgat?acaaaactcc?aggacaataa?gaaggcagtt 8580

tagaaagagt?ctctcaagaa?ctttagaaga?gtccttctac?aactcagaga?tccacgggat 8640

taatcggatg?acccagacac?ctcaaagggt?cgggagagtg?tggccttgct?cttcagagag 8700

ggcagatcta?cttagggaga?tctcttgggg?aaggaaagtg?gtaggcacga?cagttcctca 8760

cccttccgag?atgttggggc?tgcttccaaa?atcctctatt?tcctgcactt?gtggagcaac 8820

agggggaggc?aatcctagag?tttctgtatc?agtactcccg?tccttcgatc?agtcattttt 8880

ttcacgaggt?cccctaaagg?gatacttggg?ctcgtccacc?tccatgtcaa?cccagctatt 8940

ccatgcatgg?gaaaaagtca?ctaatgttca?tgtggtgaaa?agggctatat?cgttaaaaga 9000

atctataaac?tggttcatca?atagaaattc?caatttggct?caaactctaa?ttagaaacat 9060

catgtctctg?acaggccctg?atttccctct?agaagaggct?cctgttttca?aacggacagg 9120

gtcagccttg?cataggttca?agtctgccag?atacagcgaa?ggagggtatt?cttctgtttg 9180

tcctaacctt?ctctctcata?tctctgtcag?tacagacact?atgtctgatt?tgacccaaga 9240

cgggaagaac?tatgatttca?tgtttcagcc?attgatgctt?tatgcgcaaa?catggacatc 9300

agagctggta?cagagagaca?caagactgag?agactccacg?tttcactggc?accttcgatg 9360

caacagatgt?gtaaggccca?ttgaggatat?aacactggaa?acttctcaga?tcttcgagtt 9420

cccggatgtg?tcaaaaagga?tatccaggat?ggtttctgga?gccgtccctc?actttcagaa 9480

gcttcctgat?atccgtctaa?gaccaggtga?ttttgaatct?ctaagtggta?gagaaaagtc 9540

tcgccacata?gggtcagctc?aggggctctt?atactcaatc?ttagtagcaa?ttcacgattc 9600

aggatacaat?gatgggacca?tcttccctgt?caacatatac?ggcaaagttt?cccctagaga 9660

ctatttgaga?gggctcgcaa?gagggatctt?gataggatcc?tcgatttgct?tcttgacacg 9720

aatgacaaat?attaatatta?aaagacctct?tgaattgatc?tcaggggtaa?tttcctatat 9780

tctcctgagg?ctggataatc?atccctctct?gtatataatg?cttagagaac?cgtctcttag 9840

aggagagata?ttctctatcc?ctcagaaaat?ccccgccgct?tacccaacca?ctatgaaaga 9900

aggcaacaga?tcgatcttgt?gttacctcca?acacgtgcta?cgctatgagc?gagaagtaat 9960

cacggcgtcc?ccggagaatg?actggctgtg?gatcttctca?gacttcagaa?gtgcgaaaat 10020

gacgtacttg?accctcatta?cctaccagtc?tcacctccta?ctccagaggg?ttgagcgaaa 10080

cttgtctaag?agtatgagag?ctactctgcg?acaaatgggt?tccttaatga?ggcaagtgct 10140

gggtgggcac?ggagaagaca?ccttggagtc?agacgacgac?attcaacgat?tactaaaaga 10200

ctctttgcga?aggacaaggt?gggtagatca?agaggtgcgc?catgcagcta?gaaccatgag 10260

tggagattac?agccccaaca?agagagtatc?ccgcaaggca?ggatgttcag?aatgggtctg 10320

ctctgctcaa?caggttgccg?tctccacctc?agccaacccg?gcccctgtct?cagagcttga 10380

cattagggcc?ctatctaaga?ggtttcaaaa?ccccttgatc?tcgggcctaa?gagtggttca 10440

gtgggcaacc?ggcgcccatt?ataagcttaa?gcctattcta?gatgatctaa?atgttttccc 10500

atctctctgt?cttgttgttg?gagacgggtc?agggggaata?tcaagggcag?ttctcaacat 10560

gtttccagat?tctaagcttg?tgttcaacag?cctattggag?gtgaatgatc?tgatggcttc 10620

cggaacacat?ccactgcctc?cttcagcaat?catgagtgga?ggagatgata?tcatctccag 10680

agtgatagac?tttgactcaa?tctgggaaaa?accatccgac?ctgaggaact?tggccacctg 10740

gagatacttc?cagtcagttc?aaaagcaggt?caacatgtcg?tatgacctca?ttgtttgtga 10800

tgcagaagtt?actgatattg?catctatcaa?ccggataact?ctgttgatgt?ctgatttcgc 10860

attgtctata?gatggaccac?tttatctggt?cttcaaaact?tacgggacta?tgctagtgaa 10920

cccagactat?aaagctatcc?aacatctgtc?aagagcgttc?ccttcggtca?cagggtttat 10980

aacccaagta?acttcgtcct?tttcttctga?gctatacctc?cggttctcca?aacgagggaa 11040

gttttttagg?gatgctgagt?acttgacctc?ttccaccctt?cgggagatga?gccttgtgtt 11100

attcaattgt?agcagcccca?aaagtgagat?gcagagagct?cgttccttaa?actatcaaga 11160

tcttgtaagg?ggatttcctg?aagagatcat?atcaaatcct?tacaatgaaa?tgatcataac 11220

tctgattgac?agtgatgtag?agtccttcct?ggtccacaag?atggtggatg?atcttgagtt 11280

acagagggga?actctgtcta?aagtggctat?cattatatcc?atcatgattg?ttttttccaa 11340

tagagtcttc?aacatttcca?aacctttgac?tgaccccttg?ttctatcccc?catctgatcc 11400

taaaatcctg?aggcacttca?acatatgttg?cagtactatg?atgtatctat?ctaccgcttt 11460

aggcgacgtc?cctagcttcg?caagacttca?tgacctatat?aatagaccta?taacttatta 11520

cttcagaaag?caagttattc?gagggaatat?ttatctatct?tggagctggt?ccgatgacac 11580

cccagtgttc?aagagggtag?cctgtaattc?tagcttgagt?ctgtcatctc?actggatcag 11640

gttgatttac?aagatagtga?agactaccag?actcgttggc?agcatagaag?atctatcagg 11700

agaggtagaa?cgacacctgc?atgggtacaa?cagatggatc?accctcgagg?atatccgatc 11760

tagatcatcc?ctactagact?acagttgttt?gtaagccgga?cattaatgaa?agcctgtaca 11820

tgctaaaatt?cttgtatgat?gcatcttgaa?aaaaacaaga?tcttgaatcc?gaacctctgg 11880

ttgtttgatt?gtttttttta?tctttattgt?ttatttgtta?agcgt 11925

<210>2

<211>4063

<212>DNA

＜213〉rabies virus

<400>2

acgcttaaca?acaaaaccaa?agaagaagca?gacagcgtca?gttgcaaagc?aaaaatgtaa 60

cacccctaca?atggatgccg?acaagattgt?gttcaaagtc?aataatcagg?tggtctcttt 120

gaagcccgag?attatcgtgg?atcaatatga?gtacaagtac?cctgctatca?aagatttgaa 180

aaagccttgt?ataaccctag?ggaaagcccc?cgatttaaac?aaagcttaca?aatcagtttt 240

atcaggcatg?aatgccgcca?aacttgatcc?tgatgatgta?tgctcctact?tggcagcagc 300

aatgcagttc?tttgagggga?catgtccgga?agactggacc?agctatggaa?tcctgattgc 360

acgaaaagga?gacaagatca?ccccagactc?tctagtggag?ataaagcgta?ctgatgtaga 420

agggaattgg?gctctgacag?gaggcatgga?actgacaagg?gaccccactg?tctccgaaca 480

tgcatcttta?gtcggtcttc?tcctgagtct?gtacagattg?agcaaaatat?caggacaaaa 540

cactggtaac?tataagacaa?acattgcgga?tagaatagag?cagattttcg?agacagcccc 600

ttttgttaag?atcgtggaac?accataccct?gatgacaact?cacaagatgt?gtgctaattg 660

gagtactata?ccgaacttca?gatttttggc?cggaacctac?gacatgtttt?tctcacggat 720

cgagcatctg?tattcggcaa?tcagagtggg?cacagttgtc?accgcttatg?aagactgctc 780

aggactggta?tcgtttacag?ggttcataaa?gcagatcaat?ctcaccgcaa?gagaagcaat 840

actatatttc?ttccacaaga?actttgaaga?agagataagg?agaatgtttg?agccagggca 900

agagacagct?gttcctcact?cttatttcat?tcacttccgt?tcactaggct?tgagtgggaa 960

gtctccttat?tcatcaaatg?ccgtcggtca?tgtgttcaat?ctcattcact?ttgttggatg 1020

ctatatgggt?caagtcagat?ctctaaatgc?aacggttatt?gctgcatgtg?cccctcatga 1080

gatgtctgtt?ctagggggct?atttgggaga?ggagttcttc?ggaaaaggga?catttgaaag 1140

aaggttcttc?agagacgaga?aagaacttca?agaatatgag?gcggctgaac?tgacaaagac 1200

cgacgtggca?ctggcagatg?acggaaccgt?caactccgat?gacgaggact?acttctccgg 1260

tgaaaccaga?agtccagaag?ctgtctatac?tcgaatcatg?atgaatggag?gtcgactgaa 1320

gagatcacat?atacggagat?atgtctcagt?cagttccaat?catcaagccc?gtccaaactc 1380

attcgccgaa?tttttaaaca?aaacgtattc?gagtgactca?taaggagttg?aatgacaggg 1440

tgccagaaat?ccatagattg?tgtatatcca?tcatgaaaaa?aactaacact?cctcctttcg 1500

aaccatccca?agtatgagca?agatctttgt?taatccgagt?gcaatcagag?ccggtctggc 1560

cgatcttgag?atggccgaag?agactgttga?tctgatcaac?agaaacatag?aagacaatca 1620

ggctcatctc?cagggagaac?ccatagaagt?ggacaatctc?cctgaggaca?tgaggcaatt 1680

tcacctgggc?gatgaaaaat?tgtccaacct?tggtgagatg?gttagggtgg?gcgaaggcaa 1740

gtatcgagag?gactttcaga?tggatgaggg?agaggacccc?aacctcctgt?tccaatcgta 1800

cctggacaat?gttggagtcc?aaatagtcag?acaaatgagg?tcaggagaga?gattcctcaa 1860

gatatggtca?cagaccgtag?aggaaattat?atcctatgtc?acggtcaact?ttcctaaccc 1920

tccaggaagg?tcttcggagg?ataaatcaac?ccaaactact?ggccgggagc?tcaagaagga 1980

gacaacatcc?actctttctc?agagagaaag?ccaaccttca?aaagccggaa?tggtggctca 2040

agttgcctct?ggccctccat?cccttgaatg?gtctgccacc?aatgaagagg?atgatctatc 2100

agtagaggct?gagatcgctc?atcagattgc?tgaaagcttt?tccaagaagt?acaagtttcc 2160

ctctcgatct?tcaggaatat?tcttgtataa?ttttgagcaa?ctggagatga?accttgatga 2220

catagttaaa?gaggcaaaaa?atgtaccggg?cgtgacccgt?ctggcccatg?atggatccaa 2280

aatcccccta?agatgtgtac?tgggatgggt?cgctttggcc?aactccaaga?aattccaatt 2340

gatagtcgag?gccgacaagc?taagcaaaat?catgcaagat?gacttggatc?gctacacatc 2400

atgctaaccg?agtcttcgaa?ttcattccct?ctagataatg?aaaactgaga?tgtcatggag 2460

tcgacatgaa?aaaaacaggc?aacaccacta?ataaaatgaa?ctttctatgt?aagatagtga 2520

aaaactgtag?ggatgaggac?acccaaaagc?cctctcctgt?gtcagcccct?ccggatggcg 2580

atgacctgtg?gcttccacct?ccagaatatg?tcccgctgaa?agaactcaca?agcaagaaga 2640

acatgaggaa?cttttgtatc?aacggggagg?ttaaagtgtg?tagcccgaac?ggttactcat 2700

tcaggatcct?gcggcatatt?ctgagatcat?tcgacgagat?atactctggg?aatcatagga 2760

tgattgggtt?agtcaaagtt?gttattggac?tagctttatc?aggagctcca?gttcctgagg 2820

gcatgaactg?ggtatacaaa?ttgaggagaa?cccttatttt?ccagtgggct?gattccaggg 2880

gccctcttga?aggggaggag?ttggaacact?ctcaagagat?cacttgggac?gatgatactg 2940

aattcgtcgg?attgcaaatg?agagtgagcg?caagacaatg?tcatattcaa?ggcaggatct 3000

ggtgtatcaa?catgaactcg?agggcatgtc?aactatggtc?tgacatgtct?cttcagacac 3060

aaaggtctga?agaggacaaa?gactcttctc?tgcttctaga?ataatcagat?tatatcccgc 3120

aagtttatca?cttgtttacc?tctggaggag?agaacatacg?ggcttaactc?caatccttgg 3180

gagcaataga?acaaaaaaac?acgccatggt?gccattaaac?cgctgcattt?tatcaaagtc 3240

aagttaatta?cctttacatt?ttgagcctct?tggatgtgaa?aaaaactatt?aacatccctc 3300

aaaagactta?aggaaagatg?gttcctcagg?ttcttttgtt?tgtacccctc?ctgggttttt 3360

cattgtgttt?cgggaagttc?cccatttaca?cgataccaga?caaacttggt?ccctggagcc 3420

ctattgacat?acaccatctc?agctgtccaa?ataacctggt?tgtggaggac?gaaggatgta 3480

ccaacctgtc?cgagttctct?tacatggaac?ttaaagtggg?atacatctca?gccataaaag 3540

tgaacgggtt?cacttgcaca?ggtgttgtga?cagaggcaga?aacctacacc?aactttgttg 3600

gttatgtcac?aaccacattc?aagagaaagc?atttccgccc?caccccagac?gcatgtagag 3660

ccgcgtataa?ctggaagatg?gccggtgacc?ccagatatga?agagtctcta?cacaatccgt 3720

accccgacta?ccattggctt?cgaactgtaa?aaaccaccaa?agagtctctc?gttatcatat 3780

ccccaagtgt?gacagatttg?gacccatatg?acaaatccct?tcactcaagg?gtcttccctg 3840

gcggaaattg?ctcaggaata?acggtgtcct?cgacctactg?ctcaactaat?catgattaca 3900

ccatttggat?gcctgagaat?ctgagactag?ggacatcttg?tgacattttt?accaatagca 3960

gagggaagag?ggcatccaaa?ggaggcaaga?cttgcggctt?tgtggatgaa?agaggcctgt 4020

ataagtctct?aaagggagca?tgcaaactca?agttatgtgg?agt 4063

<210>3

<211>4242

<212>DNA

＜213〉rabies virus

<400>3

aggcctgtat?aagtctctaa?agggagcatg?caaactcaag?ttatgtggag?ttctcggact 60

tagacttatg?gatggaacat?gggtcgcgat?gcaaacatca?gatgagacca?aatggtgccc 120

tccaggtcag?ttggtgaatt?tgcacgactt?tcgctcagac?gagattgagc?atctcgttgt 180

ggaagagtta?gtcaagaaaa?gagaggagtg?tctggatgca?ctagagtcca?tcatgaccac 240

caagtcagtg?agtttcagac?gtctcagtca?cttgagaaaa?cttgtccctg?ggtttggaaa 300

agcatatacc?atattcaaca?aaaccttgat?ggaggctgat?gctcactaca?agtctgtccg 360

gacctggaat?gagatcatcc?cctcaaaagg?gtgtttgaga?gttgggggga?ggtgtcatcc 420

ccatgtgaac?ggggtgtttt?tcaatggtat?aatattaggg?tctgacggcc?atgttctaat 480

cccagagatg?cagtcatccc?tcctccagca?acatatggag?ttgttggaat?cttcagttat 540

ccccctgatg?caccccttgg?cagacccttc?tacagttttc?aaagacggtg?atgaggttga 600

ggattttgtt?gaagttcacc?tccccgatgt?gcatgaacag?gtctcaggag?ttgaactggg 660

tctcccgaac?tgggggaagt?atgtattgat?gattgcaggg?gccttgattg?ccctgatgtt 720

gataattttc?ctgatgacat?gttgcagaag?agtcaatcga?ccagaatcta?cgcaaagcag 780

tcttggagag?acagggagaa?atgtgtcagt?cacttcccaa?agcggaaaag?tcatatcttc 840

atgggagtca?tataagagtg?gaggcgagac?cagactgtga?aggccggtca?tccttttgac 900

acttcaagtc?ccgaggataa?cctcctctcg?gggttggggg?gaatcttggg?atccagtagt 960

cctccttgaa?ctccatccaa?cagggtagat?ttaagagtca?tgagactttc?attaatcatc 1020

tcagttgatc?agacatggtc?gtgtagattc?tcataacacg?ggagatcttc?tagcagtttc 1080

agtgaccaac?ggtgctttca?ttctccagga?actgatacca?aaggttgtgg?acaagccaag 1140

gggtgcttcg?gattactctg?tgcttgggca?cagaaagagg?tcgtagtttg?ccccttgata 1200

gcagattcaa?catgaattaa?ctaagaaagg?cgatctgcct?cccatgaagg?acataagcaa 1260

tagttcacaa?tcatcttgca?tctcagtgaa?gtgtacataa?ctataaaggg?ctgggtcatc 1320

taagcatttc?agtcgagaaa?aaaactgtag?accaaaagaa?caactaacaa?cacttctcat 1380

cccgagaccc?atatcaagat?gctggatccg?ggagaggttt?atgatgaccc?tattgatcca 1440

attgagtcag?aggctgaacc?cagaggaacc?cccactgtcc?ccaacatctt?gaggaactct 1500

gactacaatc?tcaactctcc?tttgatagag?gactctgcca?aactaatgtt?agaatggttg 1560

aaaacaggga?acagacctta?tcggatgact?ttgacagaca?attgctccag?gtcttacaaa 1620

gttttgaaag?attatttcaa?gaaagtagat?ttgggttctc?tcaaagtggg?aggaactgct 1680

gcacaatcaa?tgatttctct?ctggttgtat?ggagcccact?ctgagtcaaa?caggagccgg 1740

agatgtataa?ccgacttggc?ccatttctat?tccaagtcat?cccccataga?gaagctgttg 1800

aattgtacgt?taggaaacag?aggcctgaga?atcccaccag?agggggtgtt?aagttgcctt 1860

gagagggtcg?attatgataa?ggcatttggg?aggtatctgg?ccaacacgta?ttcctcttat 1920

ctgtttttcc?atgtaattac?cttatacatg?aatgccctag?actgggaaga?ggaaaaaacc 1980

atcctagcat?tatggaaaga?tctaacctca?gtggataccg?ggaaggactt?ggtcaaattc 2040

aaagatcaaa?tatggggact?gctggttgtg?acaaaggact?ttgtttactc?tcagagttct 2100

aactgtcttt?ttgacagaaa?ctatacactg?atgctaaagg?atcttttctt?gtctcgattc 2160

aactccttga?tgattttgct?ttctccccct?gagccccgat?actcagatga?cttaatatct 2220

cagctgtgcc?agctatacat?cgctggggat?caagtcttgt?ccttgtgtgg?gaactccggc 2280

tatgaagtca?tcaaaatatt?ggagccatat?gtcgtgaaca?gtttggtcca?gagggcagag 2340

aagtttaggc?ctctcatcca?ctccttggga?gactttccta?tgtttataaa?agacaaggtg 2400

aatcaacttg?aagggacttt?cggtcccagt?gcaaaaaggt?tttttagggt?tctagatcaa 2460

ttcgacaaca?tacatgatct?agtatttgtg?tatggctgtt?acagacattg?ggggcacccc 2520

tatatagatt?atcggaaggg?tctgtcgaaa?ctatatgatc?aagttcacat?taagaaagta 2580

atagataagt?cctaccagga?gtgtttagca?agtgacttgg?ctagaaggat?cctcagatgg 2640

ggatttgaca?agtactccaa?gtggtatcta?gattcgagat?tccttgccct?agaccacccc 2700

ttggctcctt?atatcaagac?ccaaacatgg?ccacccaaac?atatagtaga?cttggtgggg 2760

gacacatggc?acaagctccc?gatcacgcag?atctttgaga?ttcctgaatc?aatggacccg 2820

tcagagatac?tggatgataa?atcacattct?ttcaccagaa?caagactagc?ttcttggctg 2880

tccgagaacc?gaggggggcc?tgttcctagc?gagaaggtca?ttatcacggc?cctgtctaag 2940

ccacctgtca?atccccgaga?gtttttgaaa?tctatcgacc?tcggaggatt?gccagatgat 3000

gacttgataa?ttggcctcag?accaaaggaa?cgggagttga?agattgaggg?ccgattcttc 3060

gctctaatgt?catggaatct?aagattatat?tttgtcatca?ccgagaagct?cttggccaac 3120

tacattttgc?cactttttga?cgcactgact?atgacagaca?acctgaacaa?ggtatttaaa 3180

aagttgatcg?acagggtcac?cgggcaaggg?cttttggact?attcgagggt?cacatacgca 3240

tttcacctgg?actatgagaa?atggaacaat?catcaaagat?tggagtcaac?ggaggatgta 3300

ttttctgtcc?tagatcaggt?gtttggattg?aagagggtgt?tttctagaac?acacgagttt 3360

tttcagaagt?cctggatcta?ttattcagac?agatcagacc?tcattgggtt?acgggaggat 3420

cagatatatt?gcttggatat?gtccaatggt?ccaacctgct?ggaatggcca?agacggcggg 3480

ctagagggct?tacggcagaa?gggctggagt?ctagtcagct?tattgatgat?agatagagaa 3540

tctcaaacca?ggaacacaag?aaccaagata?ctagctcaag?gagacaacca?ggttctgtgt 3600

ccgacataca?tgttgtcacc?gggattgtct?caagaggggc?ttctctatga?gttagagagc 3660

atatcgagga?atgcactctc?aatataccga?gctatcgagg?aaggagcatc?taaactgggg 3720

ctgatcatca?agaaagaaga?gaccatgtgt?agttatgact?ttctcatata?tgggaagacc 3780

cccttatttc?gaggcaacat?attagtacct?gaatccaaaa?gatgggcccg?agtctcttgc 3840

atctctaacg?accaaatagt?caacctcgcc?aatataatgt?ctacagtatc?caccaatgcg 3900

ctgacagtgg?cacaacactc?tcaatctctg?atcaaaccta?tgagggattt?tctgctcatg 3960

tcagtacagg?cagttttcca?ctacctgctg?tttagcccaa?tcttaaaagg?cagggtttat 4020

aagattctga?gtgctgaagg?ggagagcttt?ctcctagcca?tgtcgcggat?aatctaccta 4080

gatccttctt?tgggaggggt?gtctggaatg?tctctcggga?ggttccatat?acgtcagttc 4140

tcagaccctg?tctctgaagg?gttgtcattc?tggagagaga?tctggttaag?ctcccatgaa 4200

tcctggattc?acgcgttgtg?tcaagaggcc?gggaaccccg?at 4242

<210>4

<211>3739

<212>DNA

＜213〉rabies virus

<400>4

gagagatctg?gttaagctcc?catgaatcct?ggattcacgc?gttgtgtcaa?gaggccggga 60

accccgatct?tggagagaga?acactagaga?gcttcactcg?ccttttagaa?gatcctacta 120

ccttaaatat?caaaggaggg?gccagtccta?ccattctact?caaggatgct?atcagaaagg 180

ctctgtacga?cgaggtggac?aaggtggaaa?attcagagtt?tcgagaggca?atcctgttgt 240

ccaagaccca?tagagataac?tttatactct?ttttaaaatc?tgttgagcct?ctgttccctc 300

gatttctcag?tgaactcttc?agttcgtcct?tcttgggaat?accagagtca?attattggac 360

tgatacaaaa?ctccaggaca?ataagaaggc?agtttagaaa?gagtctctca?agaactttag 420

aagagtcctt?ctacaactca?gagatccacg?ggattaatcg?gatgacccag?acacctcaaa 480

gggtcgggag?agtgtggcct?tgctcttcag?agagggcaga?tctacttagg?gagatctctt 540

ggggaaggaa?agtggtaggc?acgacagttc?ctcacccttc?cgagatgttg?gggctgcttc 600

caaaatcctc?tatttcctgc?acttgtggag?caacaggggg?aggcaatcct?agagtttctg 660

tatcagtact?cccgtccttc?gatcagtcat?ttttttcacg?aggtccccta?aagggatact 720

tgggctcgtc?cacctccatg?tcaacccagc?tattccatgc?atgggaaaaa?gtcactaatg 780

ttcatgtggt?gaaaagggct?atatcgttaa?aagaatctat?aaactggttc?atcaatagaa 840

attccaattt?ggctcaaact?ctaattagaa?acatcatgtc?tctgacaggc?cctgatttcc 900

ctctagaaga?ggctcctgtt?ttcaaacgga?cagggtcagc?cttgcatagg?ttcaagtctg 960

ccagatacag?cgaaggaggg?tattcttctg?tttgtcctaa?ccttctctct?catatctctg 1020

tcagtacaga?cactatgtct?gatttgaccc?aagacgggaa?gaactatgat?ttcatgtttc 1080

agccattgat?gctttatgcg?caaacatgga?catcagagct?ggtacagaga?gacacaagac 1140

tgagagactc?cacgtttcac?tggcaccttc?gatgcaacag?atgtgtaagg?cccattgagg 1200

atataacact?ggaaacttct?cagatcttcg?agttcccgga?tgtgtcaaaa?aggatatcca 1260

ggatggtttc?tggagccgtc?cctcactttc?agaagcttcc?tgatatccgt?ctaagaccag 1320

gtgattttga?atctctaagt?ggtagagaaa?agtctcgcca?catagggtca?gctcaggggc 1380

tcttatactc?aatcttagta?gcaattcacg?attcaggata?caatgatggg?accatcttcc 1440

ctgtcaacat?atacggcaaa?gtttccccta?gagactattt?gagagggctc?gcaagaggga 1500

tcttgatagg?atcctcgatt?tgcttcttga?cacgaatgac?aaatattaat?attaaaagac 1560

ctcttgaatt?gatctcaggg?gtaatttcct?atattctcct?gaggctggat?aatcatccct 1620

ctctgtatat?aatgcttaga?gaaccgtctc?ttagaggaga?gatattctct?atccctcaga 1680

aaatccccgc?cgcttaccca?accactatga?aagaaggcaa?cagatcgatc?ttgtgttacc 1740

tccaacacgt?gctacgctat?gagcgagaag?taatcacggc?gtccccggag?aatgactggc 1800

tgtggatctt?ctcagacttc?agaagtgcga?aaatgacgta?cttgaccctc?attacctacc 1860

agtctcacct?cctactccag?agggttgagc?gaaacttgtc?taagagtatg?agagctactc 1920

tgcgacaaat?gggttcctta?atgaggcaag?tgctgggtgg?gcacggagaa?gacaccttgg 1980

agtcagacga?cgacattcaa?cgattactaa?aagactcttt?gcgaaggaca?aggtgggtag 2040

atcaagaggt?gcgccatgca?gctagaacca?tgagtggaga?ttacagcccc?aacaagagag 2100

tatcccgcaa?ggcaggatgt?tcagaatggg?tctgctctgc?tcaacaggtt?gccgtctcca 2160

cctcagccaa?cccggcccct?gtctcagagc?ttgacattag?ggccctatct?aagaggtttc 2220

aaaacccctt?gatctcgggc?ctaagagtgg?ttcagtgggc?aaccggcgcc?cattataagc 2280

ttaagcctat?tctagatgat?ctaaatgttt?tcccatctct?ctgtcttgtt?gttggagacg 2340

ggtcaggggg?aatatcaagg?gcagttctca?acatgtttcc?agattctaag?cttgtgttca 2400

acagcctatt?ggaggtgaat?gatctgatgg?cttccggaac?acatccactg?cctccttcag 2460

caatcatgag?tggaggagat?gatatcatct?ccagagtgat?agactttgac?tcaatctggg 2520

aaaaaccatc?cgacctgagg?aacttggcca?cctggagata?cttccagtca?gttcaaaagc 2580

aggtcaacat?gtcgtatgac?ctcattgttt?gtgatgcaga?agttactgat?attgcatcta 2640

tcaaccggat?aactctgttg?atgtctgatt?tcgcattgtc?tatagatgga?ccactttatc 2700

tggtcttcaa?aacttacggg?actatgctag?tgaacccaga?ctataaagct?atccaacatc 2760

tgtcaagagc?gttcccttcg?gtcacagggt?ttataaccca?agtaacttcg?tccttttctt 2820

ctgagctata?cctccggttc?tccaaacgag?ggaagttttt?tagggatgct?gagtacttga 2880

cctcttccac?ccttcgggag?atgagccttg?tgttattcaa?ttgtagcagc?cccaaaagtg 2940

agatgcagag?agctcgttcc?ttaaactatc?aagatcttgt?aaggggattt?cctgaagaga 3000

tcatatcaaa?tccttacaat?gaaatgatca?taactctgat?tgacagtgat?gtagagtcct 3060

tcctggtcca?caagatggtg?gatgatcttg?agttacagag?gggaactctg?tctaaagtgg 3120

ctatcattat?atccatcatg?attgtttttt?ccaatagagt?cttcaacatt?tccaaacctt 3180

tgactgaccc?cttgttctat?cccccatctg?atcctaaaat?cctgaggcac?ttcaacatat 3240

gttgcagtac?tatgatgtat?ctatctaccg?ctttaggcga?cgtccctagc?ttcgcaagac 3300

ttcatgacct?atataataga?cctataactt?attacttcag?aaagcaagtt?attcgaggga 3360

atatttatct?atcttggagc?tggtccgatg?acaccccagt?gttcaagagg?gtagcctgta 3420

attctagctt?gagtctgtca?tctcactgga?tcaggttgat?ttacaagata?gtgaagacta 3480

ccagactcgt?tggcagcata?gaagatctat?caggagaggt?agaacgacac?ctgcatgggt 3540

acaacagatg?gatcaccctc?gaggatatcc?gatctagatc?atccctacta?gactacagtt 3600

gtttgtaagc?cggacattaa?tgaaagcctg?tacatgctaa?aattcttgta?tgatgcatct 3660

tgaaaaaaac?aagatcttga?atccgaacct?ctggttgttt?gattgttttt?tttatcttta 3720

ttgtttattt?gttaagcgt 3739

<210>5

<211>1353

<212>DNA

＜213〉rabies virus

<400>5

atggatgccg?acaagattgt?gttcaaagtc?aataatcagg?tggtctcttt?gaagcccgag 60

attatcgtgg?atcaatatga?gtacaagtac?cctgctatca?aagatttgaa?aaagccttgt 120

ataaccctag?ggaaagcccc?cgatttaaac?aaagcttaca?aatcagtttt?atcaggcatg 180

aatgccgcca?aacttgatcc?tgatgatgta?tgctcctact?tggcagcagc?aatgcagttc 240

tttgagggga?catgtccgga?agactggacc?agctatggaa?tcctgattgc?acgaaaagga 300

gacaagatca?ccccagactc?tctagtggag?ataaagcgta?ctgatgtaga?agggaattgg 360

gctctgacag?gaggcatgga?actgacaagg?gaccccactg?tctccgaaca?tgcatcttta 420

gtcggtcttc?tcctgagtct?gtacagattg?agcaaaatat?caggacaaaa?cactggtaac 480

tataagacaa?acattgcgga?tagaatagag?cagattttcg?agacagcccc?ttttgttaag 540

atcgtggaac?accataccct?gatgacaact?cacaagatgt?gtgctaattg?gagtactata 600

ccgaacttca?gatttttggc?cggaacctac?gacatgtttt?tctcacggat?cgagcatctg 660

tattcggcaa?tcagagtggg?cacagttgtc?accgcttatg?aagactgctc?aggactggta 720

tcgtttacag?ggttcataaa?gcagatcaat?ctcaccgcaa?gagaagcaat?actatatttc 780

ttccacaaga?actttgaaga?agagataagg?agaatgtttg?agccagggca?agagacagct 840

gttcctcact?cttatttcat?tcacttccgt?tcactaggct?tgagtgggaa?gtctccttat 900

tcatcaaatg?ccgtcggtca?tgtgttcaat?ctcattcact?ttgttggatg?ctatatgggt 960

caagtcagat?ctctaaatgc?aacggttatt?gctgcatgtg?cccctcatga?gatgtctgtt 1020

ctagggggct?atttgggaga?ggagttcttc?ggaaaaggga?catttgaaag?aaggttcttc 1080

agagacgaga?aagaacttca?agaatatgag?gcggctgaac?tgacaaagac?cgacgtggca 1140

ctggcagatg?acggaaccgt?caactccgat?gacgaggact?acttctccgg?tgaaaccaga 1200

agtccagaag?ctgtctatac?tcgaatcatg?atgaatggag?gtcgactgaa?gagatcacat 1260

atacggagat?atgtctcagt?cagttccaat?catcaagccc?gtccaaactc?attcgccgaa 1320

tttttaaaca?aaacgtattc?gagtgactca?taa 1353

<210>6

<211>894

<212>DNA

＜213〉rabies virus

<400>6

atgagcaaga?tctttgttaa?tccgagtgca?atcagagccg?gtctggccga?tcttgagatg 60

gccgaagaga?ctgttgatct?gatcaacaga?aacatagaag?acaatcaggc?tcatctccag 120

ggagaaccca?tagaagtgga?caatctccct?gaggacatga?ggcaatttca?cctgggcgat 180

gaaaaattgt?ccaaccttgg?tgagatggtt?agggtgggcg?aaggcaagta?tcgagaggac 240

tttcagatgg?atgagggaga?ggaccccaac?ctcctgttcc?aatcgtacct?ggacaatgtt 300

ggagtccaaa?tagtcagaca?aatgaggtca?ggagagagat?tcctcaagat?atggtcacag 360

accgtagagg?aaattatatc?ctatgtcacg?gtcaactttc?ctaaccctcc?aggaaggtct 420

tcggaggata?aatcaaccca?aactactggc?cgggagctca?agaaggagac?aacatccact 480

ctttctcaga?gagaaagcca?accttcaaaa?gccggaatgg?tggctcaagt?tgcctctggc 540

cctccatccc?ttgaatggtc?tgccaccaat?gaagaggatg?atctatcagt?agaggctgag 600

atcgctcatc?agattgctga?aagcttttcc?aagaagtaca?agtttccctc?tcgatcttca 660

ggaatattct?tgtataattt?tgagcaactg?gagatgaacc?ttgatgacat?agttaaagag 720

gcaaaaaatg?taccgggcgt?gacccgtctg?gcccatgatg?gatccaaaat?ccccctaaga 780

tgtgtactgg?gatgggtcgc?tttggccaac?tccaagaaat?tccaattgat?agtcgaggcc 840

gacaagctaa?gcaaaatcat?gcaagatgac?ttggatcgct?acacatcatg?ctaa 894

<210>7

<211>6384

<212>DNA

＜213〉rabies virus

<400>7

atgctggatc?cgggagaggt?ttatgatgac?cctattgatc?caattgagtc?agaggctgaa 60

cccagaggaa?cccccactgt?ccccaacatc?ttgaggaact?ctgactacaa?tctcaactct 120

cctttgatag?aggactctgc?caaactaatg?ttagaatggt?tgaaaacagg?gaacagacct 180

tatcggatga?ctttgacaga?caattgctcc?aggtcttaca?aagttttgaa?agattatttc 240

aagaaagtag?atttgggttc?tctcaaagtg?ggaggaactg?ctgcacaatc?aatgatttct 300

ctctggttgt?atggagccca?ctctgagtca?aacaggagcc?ggagatgtat?aaccgacttg 360

gcccatttct?attccaagtc?atcccccata?gagaagctgt?tgaattgtac?gttaggaaac 420

agaggcctga?gaatcccacc?agagggggtg?ttaagttgcc?ttgagagggt?cgattatgat 480

aaggcatttg?ggaggtatct?ggccaacacg?tattcctctt?atctgttttt?ccatgtaatt 540

accttataca?tgaatgccct?agactgggaa?gaggaaaaaa?ccatcctagc?attatggaaa 600

gatctaacct?cagtggatac?cgggaaggac?ttggtcaaat?tcaaagatca?aatatgggga 660

ctgctggttg?tgacaaagga?ctttgtttac?tctcagagtt?ctaactgtct?ttttgacaga 720

aactatacac?tgatgctaaa?ggatcttttc?ttgtctcgat?tcaactcctt?gatgattttg 780

ctttctcccc?ctgagccccg?atactcagat?gacttaatat?ctcagctgtg?ccagctatac 840

atcgctgggg?atcaagtctt?gtccttgtgt?gggaactccg?gctatgaagt?catcaaaata 900

ttggagccat?atgtcgtgaa?cagtttggtc?cagagggcag?agaagtttag?gcctctcatc 960

cactccttgg?gagactttcc?tatgtttata?aaagacaagg?tgaatcaact?tgaagggact 1020

ttcggtccca?gtgcaaaaag?gttttttagg?gttctagatc?aattcgacaa?catacatgat 1080

ctagtatttg?tgtatggctg?ttacagacat?tgggggcacc?cctatataga?ttatcggaag 1140

ggtctgtcga?aactatatga?tcaagttcac?attaagaaag?taatagataa?gtcctaccag 1200

gagtgtttag?caagtgactt?ggctagaagg?atcctcagat?ggggatttga?caagtactcc 1260

aagtggtatc?tagattcgag?attccttgcc?ctagaccacc?ccttggctcc?ttatatcaag 1320

acccaaacat?ggccacccaa?acatatagta?gacttggtgg?gggacacatg?gcacaagctc 1380

ccgatcacgc?agatctttga?gattcctgaa?tcaatggacc?cgtcagagat?actggatgat 1440

aaatcacatt?ctttcaccag?aacaagacta?gcttcttggc?tgtccgagaa?ccgagggggg 1500

cctgttccta?gcgagaaggt?cattatcacg?gccctgtcta?agccacctgt?caatccccga 1560

gagtttttga?aatctatcga?cctcggagga?ttgccagatg?atgacttgat?aattggcctc 1620

agaccaaagg?aacgggagtt?gaagattgag?ggccgattct?tcgctctaat?gtcatggaat 1680

ctaagattat?attttgtcat?caccgagaag?ctcttggcca?actacatttt?gccacttttt 1740

gacgcactga?ctatgacaga?caacctgaac?aaggtattta?aaaagttgat?cgacagggtc 1800

accgggcaag?ggcttttgga?ctattcgagg?gtcacatacg?catttcacct?ggactatgag 1860

aaatggaaca?atcatcaaag?attggagtca?acggaggatg?tattttctgt?cctagatcag 1920

gtgtttggat?tgaagagggt?gttttctaga?acacacgagt?tttttcagaa?gtcctggatc 1980

tattattcag?acagatcaga?cctcattggg?ttacgggagg?atcagatata?ttgcttggat 2040

atgtccaatg?gtccaacctg?ctggaatggc?caagacggcg?ggctagaggg?cttacggcag 2100

aagggctgga?gtctagtcag?cttattgatg?atagatagag?aatctcaaac?caggaacaca 2160

agaaccaaga?tactagctca?aggagacaac?caggttctgt?gtccgacata?catgttgtca 2220

ccgggattgt?ctcaagaggg?gcttctctat?gagttagaga?gcatatcgag?gaatgcactc 2280

tcaatatacc?gagctatcga?ggaaggagca?tctaaactgg?ggctgatcat?caagaaagaa 2340

gagaccatgt?gtagttatga?ctttctcata?tatgggaaga?cccccttatt?tcgaggcaac 2400

atattagtac?ctgaatccaa?aagatgggcc?cgagtctctt?gcatctctaa?cgaccaaata 2460

gtcaacctcg?ccaatataat?gtctacagta?tccaccaatg?cgctgacagt?ggcacaacac 2520

tctcaatctc?tgatcaaacc?tatgagggat?tttctgctca?tgtcagtaca?ggcagttttc 2580

cactacctgc?tgtttagccc?aatcttaaaa?ggcagggttt?ataagattct?gagtgctgaa 2640

ggggagagct?ttctcctagc?catgtcgcgg?ataatctacc?tagatccttc?tttgggaggg 2700

gtgtctggaa?tgtctctcgg?gaggttccat?atacgtcagt?tctcagaccc?tgtctctgaa 2760

gggttgtcat?tctggagaga?gatctggtta?agctcccatg?aatcctggat?tcacgcgttg 2820

tgtcaagagg?ccgggaaccc?cgatcttgga?gagagaacac?tagagagctt?cactcgcctt 2880

ttagaagatc?ctactacctt?aaatatcaaa?ggaggggcca?gtcctaccat?tctactcaag 2940

gatgctatca?gaaaggctct?gtacgacgag?gtggacaagg?tggaaaattc?agagtttcga 3000

gaggcaatcc?tgttgtccaa?gacccataga?gataacttta?tactcttttt?aaaatctgtt 3060

gagcctctgt?tccctcgatt?tctcagtgaa?ctcttcagtt?cgtccttctt?gggaatacca 3120

gagtcaatta?ttggactgat?acaaaactcc?aggacaataa?gaaggcagtt?tagaaagagt 3180

ctctcaagaa?ctttagaaga?gtccttctac?aactcagaga?tccacgggat?taatcggatg 3240

acccagacac?ctcaaagggt?cgggagagtg?tggccttgct?cttcagagag?ggcagatcta 3300

cttagggaga?tctcttgggg?aaggaaagtg?gtaggcacga?cagttcctca?cccttccgag 3360

atgttggggc?tgcttccaaa?atcctctatt?tcctgcactt?gtggagcaac?agggggaggc 3420

aatcctagag?tttctgtatc?agtactcccg?tccttcgatc?agtcattttt?ttcacgaggt 3480

cccctaaagg?gatacttggg?ctcgtccacc?tccatgtcaa?cccagctatt?ccatgcatgg 3540

gaaaaagtca?ctaatgttca?tgtggtgaaa?agggctatat?cgttaaaaga?atctataaac 3600

tggttcatca?atagaaattc?caatttggct?caaactctaa?ttagaaacat?catgtctctg 3660

acaggccctg?atttccctct?agaagaggct?cctgttttca?aacggacagg?gtcagccttg 3720

cataggttca?agtctgccag?atacagcgaa?ggagggtatt?cttctgtttg?tcctaacctt 3780

ctctctcata?tctctgtcag?tacagacact?atgtctgatt?tgacccaaga?cgggaagaac 3840

tatgatttca?tgtttcagcc?attgatgctt?tatgcgcaaa?catggacatc?agagctggta 3900

cagagagaca?caagactgag?agactccacg?tttcactggc?accttcgatg?caacagatgt 3960

gtaaggccca?ttgaggatat?aacactggaa?acttctcaga?tcttcgagtt?cccggatgtg 4020

tcaaaaagga?tatccaggat?ggtttctgga?gccgtccctc?actttcagaa?gcttcctgat 4080

atccgtctaa?gaccaggtga?ttttgaatct?ctaagtggta?gagaaaagtc?tcgccacata 4140

gggtcagctc?aggggctctt?atactcaatc?ttagtagcaa?ttcacgattc?aggatacaat 4200

gatgggacca?tcttccctgt?caacatatac?ggcaaagttt?cccctagaga?ctatttgaga 4260

gggctcgcaa?gagggatctt?gataggatcc?tcgatttgct?tcttgacacg?aatgacaaat 4320

attaatatta?aaagacctct?tgaattgatc?tcaggggtaa?tttcctatat?tctcctgagg 4380

ctggataatc?atccctctct?gtatataatg?cttagagaac?cgtctcttag?aggagagata 4440

ttctctatcc?ctcagaaaat?ccccgccgct?tacccaacca?ctatgaaaga?aggcaacaga 4500

tcgatcttgt?gttacctcca?acacgtgcta?cgctatgagc?gagaagtaat?cacggcgtcc 4560

ccggagaatg?actggctgtg?gatcttctca?gacttcagaa?gtgcgaaaat?gacgtacttg 4620

accctcatta?cctaccagtc?tcacctccta?ctccagaggg?ttgagcgaaa?cttgtctaag 4680

agtatgagag?ctactctgcg?acaaatgggt?tccttaatga?ggcaagtgct?gggtgggcac 4740

ggagaagaca?ccttggagtc?agacgacgac?attcaacgat?tactaaaaga?ctctttgcga 4800

aggacaaggt?gggtagatca?agaggtgcgc?catgcagcta?gaaccatgag?tggagattac 4860

agccccaaca?agagagtatc?ccgcaaggca?ggatgttcag?aatgggtctg?ctctgctcaa 4920

caggttgccg?tctccacctc?agccaacccg?gcccctgtct?cagagcttga?cattagggcc 4980

ctatctaaga?ggtttcaaaa?ccccttgatc?tcgggcctaa?gagtggttca?gtgggcaacc 5040

ggcgcccatt?ataagcttaa?gcctattcta?gatgatctaa?atgttttccc?atctctctgt 5100

cttgttgttg?gagacgggtc?agggggaata?tcaagggcag?ttctcaacat?gtttccagat 5160

tctaagcttg?tgttcaacag?cctattggag?gtgaatgatc?tgatggcttc?cggaacacat 5220

ccactgcctc?cttcagcaat?catgagtgga?ggagatgata?tcatctccag?agtgatagac 5280

tttgactcaa?tctgggaaaa?accatccgac?ctgaggaact?tggccacctg?gagatacttc 5340

cagtcagttc?aaaagcaggt?caacatgtcg?tatgacctca?ttgtttgtga?tgcagaagtt 5400

actgatattg?catctatcaa?ccggataact?ctgttgatgt?ctgatttcgc?attgtctata 5460

gatggaccac?tttatctggt?cttcaaaact?tacgggacta?tgctagtgaa?cccagactat 5520

aaagctatcc?aacatctgtc?aagagcgttc?ccttcggtca?cagggtttat?aacccaagta 5580

acttcgtcct?tttcttctga?gctatacctc?cggttctcca?aacgagggaa?gttttttagg 5640

gatgctgagt?acttgacctc?ttccaccctt?cgggagatga?gccttgtgtt?attcaattgt 5700

agcagcccca?aaagtgagat?gcagagagct?cgttccttaa?actatcaaga?tcttgtaagg 5760

ggatttcctg?aagagatcat?atcaaatcct?tacaatgaaa?tgatcataac?tctgattgac 5820

agtgatgtag?agtccttcct?ggtccacaag?atggtggatg?atcttgagtt?acagagggga 5880

actctgtcta?aagtggctat?cattatatcc?atcatgattg?ttttttccaa?tagagtcttc 5940

aacatttcca?aacctttgac?tgaccccttg?ttctatcccc?catctgatcc?taaaatcctg 6000

aggcacttca?acatatgttg?cagtactatg?atgtatctat?ctaccgcttt?aggcgacgtc 6060

cctagcttcg?caagacttca?tgacctatat?aatagaccta?taacttatta?cttcagaaag 6120

caagttattc?gagggaatat?ttatctatct?tggagctggt?ccgatgacac?cccagtgttc 6180

aagagggtag?cctgtaattc?tagcttgagt?ctgtcatctc?actggatcag?gttgatttac 6240

aagatagtga?agactaccag?actcgttggc?agcatagaag?atctatcagg?agaggtagaa 6300

cgacacctgc?atgggtacaa?cagatggatc?accctcgagg?atatccgatc?tagatcatcc 6360

ctactagact?acagttgttt?gtaa 6384

<210>8

<211>1801

<212>DNA

＜213〉rabies virus

<400>8

aagtttatca?cttgtttacc?tctggaggag?agaacatacg?ggcttaactc?caatccttgg 60

gagcaataga?acaaaaaaac?acgccatggt?gccattaaac?cgctgcattt?tatcaaagtc 120

aagttaatta?cctttacatt?ttgagcctct?tggatgtgaa?aaaaactatt?aacatccctc 180

aaaagactta?aggaaagatg?gttcctcagg?ttcttttgtt?tgtacccctc?ctgggttttt 240

cattgtgttt?cgggaagttc?cccatttaca?cgataccaga?caaacttggt?ccctggagcc 300

ctattgacat?acaccatctc?agctgtccaa?ataacctggt?tgtggaggac?gaaggatgta 360

ccaacctgtc?cgagttctct?tacatggaac?ttaaagtggg?atacatctca?gccataaaag 420

tgaacgggtt?cacttgcaca?ggtgttgtga?cagaggcaga?aacctacacc?aactttgttg 480

gttatgtcac?aaccacattc?aagagaaagc?atttccgccc?caccccagac?gcatgtagag 540

ccgcgtataa?ctggaagatg?gccggtgacc?ccagatatga?agagtctcta?cacaatccgt 600

accccgacta?ccattggctt?cgaactgtaa?aaaccaccaa?agagtctctc?gttatcatat 660

ccccaagtgt?gacagatttg?gacccatatg?acaaatccct?tcactcaagg?gtcttccctg 720

gcggaaattg?ctcaggaata?acggtgtcct?cgacctactg?ctcaactaat?catgattaca 780

ccatttggat?gcctgagaat?ctgagactag?ggacatcttg?tgacattttt?accaatagca 840

gagggaagag?ggcatccaaa?ggaggcaaga?cttgcggctt?tgtggatgaa?agaggcctgt 900

ataagtctct?aaagggagca?tgcaaactca?agttatgtgg?agttctcgga?cttagactta 960

tggatggaac?atgggtcgcg?atgcaaacat?cagatgagac?caaatggtgc?cctccaggtc 1020

agttggtgaa?tttgcacgac?tttcgctcag?acgagattga?gcatctcgtt?gtggaagagt 1080

tagtcaagaa?aagagaggag?tgtctggatg?cactagagtc?catcatgacc?accaagtcag 1140

tgagtttcag?acgtctcagt?cacttgagaa?aacttgtccc?tgggtttgga?aaagcatata 1200

ccatattcaa?caaaaccttg?atggaggctg?atgctcacta?caagtctgtc?cggacctgga 1260

atgagatcat?cccctcaaaa?gggtgtttga?gagttggggg?gaggtgtcat?ccccatgtga 1320

acggggtgtt?tttcaatggt?ataatattag?ggtctgacgg?ccatgttcta?atcccagaga 1380

tgcagtcatc?cctcctccag?caacatatgg?agttgttgga?atcttcagtt?atccccctga 1440

tgcacccctt?ggcagaccct?tctacagttt?tcaaagacgg?tgatgaggtt?gaggattttg 1500

ttgaagttca?cctccccgat?gtgcatgaac?aggtctcagg?agttgaactg?ggtctcccga 1560

actgggggaa?gtatgtattg?atgattgcag?gggccttgat?tgccctgatg?ttgataattt 1620

tcctgatgac?atgttgcaga?agagtcaatc?gaccagaatc?tacgcaaagc?agtcttggag 1680

agacagggag?aaatgtgtca?gtcacttccc?aaagcggaaa?agtcatatct?tcatgggagt 1740

catataagag?tggaggcgag?accagactgt?gaaggccggt?catccttttg?acacttcaag 1800

t 1801

Claims (10)

1. one kind has better security and immunogenic recombinant rabies virus strain, and described virus strain is characterised in that the extra G gene of insertion in its genome.

2. the recombinant rabies virus strain of claim 1, it comprises two G genes in genome.

3. the recombinant rabies virus strain of claim 1, described recombinant virus are to be made up by the Flury-LEP of wild-type rabies virus strain.

4. the recombinant rabies virus strain of claim 1, the sequence of the G gene of described insertion is shown in SEQID NO:8.

5. one kind makes up each the method for recombinant rabies virus strain of claim 1-4, and described method comprises the following steps;

1) structure is transcribed plasmid, wherein the G gene order is inserted in the genome of wild-type rabies virus strain, obtains containing the recombination group full length cDNA clone of two G genes;

2) make up the helper plasmid system, nucleoprotein N, phosphoric acid albumen P and the polymerase protein L of the described helper plasmid difference encoding wild type LEP of rabies virus strain of system;

3) use the plasmid vector of the recombination group full length cDNA clone contain two G genes and helper plasmid cotransfection to the host cell of allowing described virus replication, cultivate described host cell;

4) rescue virus.

6. the method for claim 5, wherein the plasmid of transcribing of Gou Jianing is pCI-LEP-G, the helper plasmid of structure is respectively pCAGG-N, pCAGG-P, pCAGG-L.

7. claim 5 or 6 method, wherein used host cell is the BHK-21 cell.

8. a plasmid vector wherein contains each constructed recombination group full length cDNA clone that contains two G genes just like claim 1-4.

9. the plasmid vector of claim 8, described plasmid is pCI.

10. the application that is used to prepare the rabies virus inactivated vaccine as the recombinant virus of each structure of claim 1-4.

Images