CN104630160B - Express the recombinant Newcastle disease LaSota vaccine strains of west nile fever virus PrM/E albumen - Google Patents

Abstract

The present invention successfully constructs expression west nile fever virus (West Nile virus, WNV) the recombinant Newcastle disease virus LaSota vaccine strain rLa WNV PrM/E of PrM/E albumen, its preserving number is CGMCC No.10199, and the humoral and cellular immune response reaction that system evaluation recombinant virus produces in mouse model.The results show that the vaccine strain can correctly express PrM/E albumen.C57BL/6 mouse are immunized in recombinant virus twice, ELISA is the result shows that immune mouse can produce high-caliber PrM/E protein-specifics IgG；Mouse, which is immunized, in neutralization test the results show can produce high-caliber WNV neutralizing antibodies；Flow cytometry shows that immune mouse can produce WNVE protein epitope specific C D4+ and CD8+T cell immune responses.The vaccine strain has highly important strategic importance as a kind of forward-looking and deposit property, safe and efficient West Nile fever prevention and control candidate vaccine, potential threat that should be sick to China.

Description

Express the recombinant Newcastle disease LaSota vaccine strains of west nile fever virus PrM/E albumen

Technical field

It is new that the present invention provides a kind of restructuring of expression west nile fever virus (West Nile virus, WNV) PrM/E albumen City epidemic disease poison LaSota vaccine strains and its application.Specifically, the present invention provides the weight of expression west nile fever virus PrM/E albumen Organize newcastle disease virus LaSota vaccine strains rLa-WNV-PrM/E (its preserving number is CGMCC No.10199) and its be used in preparation Prevent the application in the vaccine of West Nile fever.

Background technology

West nile virus (West Nile Virus, WNV) is one kind by the mosquito-borne arboviruse in storehouse.WNV belongs to jaundice Malicious section's Flavivirus, is distributed widely in Africa, the European west and south, Russia, the Middle East, India and Australia.The virus in Incoming North America [1,2] in 1999.West nile virus belongs to encephalitis B sero-group in serology, and the virus with group also has holy road This easy encephalitis viruses, Murray valley encephalitis virus, japanese encephalitis virus and elder brother Tianjin virus.Israel is from the stork of morbidity within 1998 It is WNV viruses to be separated to 1, its caused clinical symptoms is encephalitis and paralysis.1999, WNV epidemic situations occurred for New York, separate To NY99 strains 1 be WNV viral [3].U.S. first time WNV seriously broke out generation in 2003, and 9000 people infect, and dead 265 People.Second of serious outburst occurred in 2012, and 5387 people infect, and 243 people are dead.With explosion facies ratio in 2003, feel within 2012 The case ratio that nervous symptoms are presented in dye crowd is substantially increased.West Nile fever in 2012 is in Italy, Israel, Russia, There are generation in Serbia, Tunisia and Greece, amount to 167 [4].

West nile fever virus host range is wider, can infect people, horse, dog, sheep, alpaca etc. [5,6], most of animal Infected in stealth, clinical symptoms are presented in only a few.Most people infect west nile fever virus and only cause mild symptoms or be not true to type Clinical symptoms, show as high fever, fash, headache, aching pain of muscles etc.；A minority develops into serious lethal neurosis Shape, such as encephalitis, encephalomyelitis and relevant complication, the sick incubation period are generally 3-12 days [7,8].Birds, especially Crow is the important reservoir of WNV, and Migrants play an important role [3] during virus is disseminated.HAEMATOPHAGOUS ARTHROPODS is such as Mosquito, sand fly, midge, tick etc. are the main biographies that the mosquito such as the communication media of west nile virus, culex, yellow-fever mosquito, anopheles is the disease Medium is broadcast, the northern house of wherein American continent is the main communication media in America.Confirm WNV in mosquito with being followed in birds Ring.The mankind mainly infect west nile virus [9] by viruliferous bite by mosquitos.

The polyprotein of WNV genomic expressions, is cut by protease and produces three structural proteins C, PrM and E protein and 7 A non-structural protein.C protein is the main component of virus nucleocapsid.PrM is virocyte film anchorin and viral phase The precursor protein of the M albumen of pass.PrM protein carboxyl terminals contain one section of trans-membrane region, which can assist PrM albumen and cell Film combination, while can also be as the signal peptide of E protein, while study confirmation PrM albumen and the close phase of correctly folding of E protein Close [10-12].In addition, PrM can combine to form heterodimeric body protein with E protein and then be assembled into immature virion [13].When virus is from intracellular release, can be cut positioned at the furin protease of Golgi complex and other protease It is cut into as ripe M albumen, so as to form the virion [14] of maturation.The E protein of WNV is its most important structural proteins, It plays a key role in multiple links of virus replicative cycle, its function include acceptor combine, film fusion, virus assembly and Budding release etc..E protein is the main structural proteins [15,16] for inducing body to produce neutralizing antibody.E protein can also induce CTL killer T cells immune response [17] with immune protection effectiveness.

West Nile fever is a kind of important Amphixenosis, and China not yet finds west Nile encephalitis case, do not have at present It is separated to west nile virus.In recent years, with international personnel and goods and materials flow faster, the infected, the malicious livestock and poultry of band and medium mosquito The possibility that worm is passed to China increasingly increases.In addition west nile virus is mainly distributed on the temperature of 66.50 ° of 23.50 °-south latitude of north latitude Band area, and China major part territory is in this area, and have suitable birds host, susceptible animal and Mosquito Vectors point Cloth, therefore it is faced with west nile virus input and popular threat.Thus, there is an urgent need for develop a kind of forward-looking and deposit Property, safe and efficient West Nile fever prevention and control candidate vaccine.

The content of the invention

The purpose of the present invention is exploitation is a kind of forward-looking and deposit property, safe and efficient West Nile fever prevention and control candidate's epidemic disease Seedling.

To achieve the above object, the present inventor utilizes recombinant Newcastle disease virus reverse genetic operating system, constructs expression The recombinant Newcastle disease virus of west nile fever virus PrM/E albumen, experiment show that the recombinant virus is safe and effective in mouse, can The neutralizing antibody and E protein specific C D4+ and CD8+T cell immune response of induced high levels.Result of study shows restructuring disease Poison is outstanding West Nile fever deposit vaccine, and the sick threat is tackled to China has its own strategic significance.

Therefore, the present invention provides a kind of weight of expression west nile fever virus (West Nile virus, WNV) PrM/E albumen Group newcastle disease virus LaSota vaccine strains and its preparation method and application.Specifically, the present invention provides expression West Nile fever disease (its preserving number is CGMCC to the recombinant Newcastle disease virus LaSota vaccine strains rLa-WNV-PrM/E of malicious PrM/E albumen No.10199) and its application in being used to prevent the vaccine of West Nile fever is being prepared.

Specifically, the present invention successfully construct expression west nile fever virus PrM/E albumen (SEQ ID No.2) restructuring it is new City epidemic disease poison LaSota vaccine strain rLa-WNV-PrM/E, the vaccine strain are preserved in China Microbiological on December 12nd, 2014 Culture presevation administration committee common micro-organisms center (CGMCC, city of BeiJing, China Chaoyang District North Star West Road 1 institute 3, China Institute of microbiology of the academy of sciences, 100101), preserving number is CGMCC No.10199, and the system evaluation institute in mouse model State the humoral and cellular immune response reaction of recombinant virus generation.

The present inventor constructs the restructuring disease of expression WNV PrM/E albumen using newcastle disease virus reverse genetic operating system Malicious newcastle disease virus rLa-WNV-PrM/E.The expression of RT-PCR and indirect immunofluorescence experiment detection recombinant protein；Recombinant virus Immune mouse, with enzyme-linked immunosorbent assay (ELISA) and the humoral immunity water of WNV plaque reduction assays detection recombinant virus It is flat；The specific cellular immunity produced with flow cytometry evaluation recombinant virus is horizontal.The results show that the vaccine strain can be correct Express PrM/E albumen.C57BL/6 mouse are immunized in recombinant virus twice, ELISA is the result shows that immune mouse can produce high level PrM/E protein-specifics IgG；Mouse, which is immunized, in neutralization test the results show can produce high-caliber WNV neutralizing antibodies；Streaming Cell art shows that immune mouse can produce WNV E protein epitope specificity CD4+ and CD8+T cell immune responses.

West nile fever virus PrM/E genes used pass through the PrM/E genes to NY99 plants of WNV in the present invention (Genbank registration numbers are DQ211652.1) is synthesized into after carrying out mammalian codons optimization, the sequence after optimization For SEQ ID No.1, the amino acid sequence of its PrM/E albumen encoded is as shown in SEQ ID No.2.

The present invention also provides the recombinant Newcastle disease virus LaSota vaccine strains rLa- of expression west nile fever virus PrM/E albumen WNV-PrM/E is being prepared for the application in the vaccine of prevention disease as caused by west nile fever virus.

Preferably, the present invention provides a kind of vaccine for being used to prevent the disease as caused by west nile fever virus, and it includes have The recombinant Newcastle disease virus LaSota vaccine strains rLa- of the expression west nile fever virus PrM/E albumen of the present invention of effect amount WNV-PrM/E.The subject that vaccine progress prevention effect can be applied is mainly mammal, such as, but not limited to, people, Horse, dog, sheep, alpaca, mouse, rat etc..

The disease as caused by west nile fever virus is such as, but not limited to, high fever, fash, headache, aching pain of muscles, severe patient Such as encephalitis, encephalomyelitis and relevant complication etc..

The present invention also provides the method for preventing the disease as caused by west nile fever virus in mammal, the method Restructuring new city including applying a effective amount of expression west nile fever virus PrM/E albumen comprising the present invention to the mammal The vaccine of epidemic disease poison LaSota vaccine strains rLa-WNV-PrM/E.

The recombinant Newcastle disease virus LaSota vaccine strains rLa- of expression west nile fever virus PrM/E albumen of the present invention WNV-PrM/E can also carry out combined immunization with other vaccine strains.

In conclusion the present invention provides following embodiments：

1. the recombinant Newcastle disease virus of one kind expression west nile fever virus (West Nile virus) PrM/E albumen LaSota vaccine strains, wherein the amino acid sequence of the PrM/E albumen is as shown in SEQ ID No.2.

2. the recombinant Newcastle disease virus LaSota vaccine strains according to the 1st, it is named as rLa-WNV-PrM/E, protects Tibetan number is CGMCC No.10199.

3. a kind of vaccine for being used to prevent the disease as caused by west nile fever virus in mammal, it includes a effective amount of The recombinant Newcastle disease virus LaSota vaccine strains rLa- of expression west nile fever virus PrM/E albumen described in 1st or the 2nd WNV-PrM/E。

4. according to the vaccine described in the 3rd, wherein the mammal selects white man, horse, dog, sheep, alpaca, mouse or big Mouse.

5. according to the vaccine described in the 4th, wherein the mammal is behaved.

6. according to the vaccine described in the 3rd, wherein the disease as caused by the west nile fever virus is selected from by West Nile fever High fever, fash, headache, aching pain of muscles, encephalitis, encephalomyelitis or relevant complication caused by virus.

7. the recombinant Newcastle disease virus LaSota epidemic diseases of the expression west nile fever virus PrM/E albumen described in the 1st or the 2nd Seedling strain rLa-WNV-PrM/E is being prepared for preventing answering in the vaccine of the disease as caused by west nile fever virus in mammal With.

8. according to the application described in the 7th, wherein the mammal selects white man, horse, dog, sheep, alpaca, mouse or big Mouse.

9. according to the application described in the 7th, wherein the mammal is behaved.

10. according to the application described in the 7th, wherein the disease as caused by the west nile fever virus is selected from by West Nile fever High fever, fash, headache, aching pain of muscles, encephalitis, encephalomyelitis or relevant complication caused by virus.

11. a kind of method for preventing the disease as caused by west nile fever virus in mammal, the described method includes to institute State the vaccine that mammal applies a effective amount of 3rd.

Brief description of the drawings

From detailed description below in conjunction with the accompanying drawings, features described above of the invention and advantage will be apparent from, wherein：

The structure signal of the recombinant virus newcastle disease virus rLa-WNV-PrM/E of Fig. 1 display expression WNV PrM/E albumen Figure；

Fig. 2 shows the RT-PCR qualification results of recombinant virus.Swimming lane 1：Control；Swimming lane 2：Purpose band, molecular weight are about 2000bp；Swimming lane M：DL5000 molecular weight markers；

Fig. 3 shows the indirect immunofluorescence of recombinant virus rLa-WNV-PrM/E infection cells.(a) E protein monoclonal antibody Mark, (b) newcastle epidemic disease antibody mark, (c) PrM protein monoclonal antibodies mark and (d) negative control；

Fig. 4 shows expression and positioning of the WNV E proteins in the cell of infection.Lastrow represents rLa-WNV-PrM/ infection The immunostaining of cell, represents the primary antibody order added by immunofluorescence, is followed successively by successively from left to right：The anti-newcastle epidemic disease antibody of chicken, Mouse source WNV E protein monoclonal antibodies, DAPI (4 ', 6- diamidino -2-phenylindone (4 ', 6-diamidino-2- Phenylindole), a kind of dyestuff for marking nucleus), Merged represents the superposition of first three width image, the results show restructuring Vaccine can be starched in infection cell and the E protein of cell membrane surface great expression WNV；Next line represents rLa infection cell conducts Control, represents same lastrow, the results show parent plant rLa does not express E protein, i.e., cannot be by the list of E protein successively from left to right Anti-dye；

Fig. 5 shows the chicken embryo growth kinetics of recombinant virus rLa-WNV-PrM/E；

Fig. 6 shows securities of the recombinant virus rLa-WNV-PrM/E to mouse；

Fig. 7 shows that the ELISA antibody levels of mouse are immunized in recombinant virus rLa-WNV-PrM/E；

Fig. 8 shows that the neutralizing antibody of recombinant virus rLa-WNV-PrM/E immune serums is horizontal；

Fig. 9 shows that the E protein epitope specificity CD4+T cellular immunities (a) of mouse are immunized in recombinant virus rLa-WNV-PrM/E With CD8+T cellular immunities (b).

Embodiment

The present invention is further described referring to specific embodiment, it will be appreciated by those skilled in the art that this hair It is bright to be not limited to these specific embodiments.

1. materials and methods

1.1.1：Strain and plasmid：Newcastle disease virus LaSota vaccine strains (GenBank accession number AY845400.2), new city Epidemic disease LaSota vaccine strain reverse genetic operating systems are prepared by this laboratory, and preparation method is referring to the patent for having obtained mandate：Shen Please number：200510097997.8 (entitled " ND LaSota vaccine strain reverse genetic operating system and its application ", hair A person of good sense：Chigo is walked, ageing is blue, 2005 application times September 2 days), the restructuring acne disease vTF7-3 of T7 polymerases is expressed by the U.S. Bernard doctors Moss of NIH provide, and prepare and are carried out with titration process by document [18], -70 DEG C of guarantors of virus liquid after titration Deposit spare.LaSota full-length genomes transcribe plasmid pBRN-FL and helper plasmid pBSNP, pBSP, pBSL by this laboratory system It is standby and preserve [referring to document 19].(Genbank registration numbers are PrM/E genes containing NY99 plants of codon optimizations of WNV DQ211652.1, is synthesized into after carrying out mammalian codons optimization using bioinformatics software) by Nanjing JaRa biology Engineering company's synthesis (sequence of mammalian codons optimization is SEQ ID No.1), and it is cloned into commercial plasmid vector pUC The multiple cloning sites of 57 (purchasing white Thermo Scientific), are named as pUC57-WNV-PrM/E.

1.1.2：Cell and serum：BHK-21 cells (purchase white ATCC American type culture collections, preserving number ATCC CCL-10)) preserved by this laboratory.BHK-21 cell growths and maintaining liquid are the DMEM (Gibco) containing 5% hyclone.

1.1.3 monoclonal antibody, polypeptide, WNV antigens：West nile virus E protein monoclonal antibody by this laboratory according to Prepared by standard molecular biology method preserves；West nile fever virus E protein CD4+T cell epitopes E641 (PVGRLVTVNPFVSVA), CD8+T cell epitopes E294 (LGMSNRDFL, H2D^b) synthesized by Asia Optical of BeiJing ZhongKe and by This laboratory preserves.The west nile fever virus PrM/E albumen of purifying is prepared by this laboratory according to standard molecular biology method Preserve, antigen is detected as ELISA.

1.1.4：Experimental animal：6 week old female C57BL/6 mouse purchase experimental animal company of white Beijing dimension tonneau China.

1.2：Express the structure of the restructuring NDV genome cDNAs of WNV PrM/E albumen

Using pUC57-WNV-PrM/E as template, held by PCR primer in PrM/E gene opens reading frame 5 ' and introduce Pme I Restriction enzyme site identification sequence (GTTTAAAC) and the transcription terminator GE of NDV self-polymerization enzymes L identifications (TTAAGAAAAAA) and transcriptional initiation sequence GS (ACGGGTAGAA) Pme I restriction enzyme sites, are introduced at 3 ' ends；PCR is produced For thing after sequence verification, pBRN-FL-PmeI is inserted into end after Pme I digestions, the recombinant full-lenght genome cDNA gram being built into It is grand to be named as pBRN-FL-PrM/E.As shown in SEQ ID No.1, it is encoded wherein WNV PrM/E gene opens reading frame sequence Albumen amino acid sequence as shown in SEQ ID No.2.

Table 1. expresses the primer used in WNV PrM/E full length protein genomic clone plasmid constructions

Note：Overstriking font：Pme I sites；Lowercase：NDV genes originate and terminator sequence；Italic：Kozak sequences； Underscore WNV PrM/E gene orders

1.3：The rescue of recombinant virus

Six orifice plate of BHK-21 cell inoculations, when cell growth reaches 80% and converges, infects poxvirus by MOI=0.01 VTF7-3, infection 1 it is small when after with calcium phosphate transfection method by full-length genome plasmid pBRN-FL-PrM/E and helper plasmid pBS-N, PBS-P and pBS-L respectively with 5 μ g, 2.5 μ g, 1.25 μ g, 1.25 μ g ratio cotransfection BHK-21 cells.When transfection 8-12 is small Afterwards, transfection supernatant is discarded, with the PBS buffer shock cell 2.5 minutes containing 10%DMSO, adds complete DMEM nutrient solutions afterwards (purchasing white Gibco), 12 it is small when after replace Opi-MEM culture mediums (purchasing white Gibco), and add TPCK processing pancreatin (1 μ g/mL, Purchase white Sigma) 37 DEG C continue to be incubated 72 it is small when after harvest culture supernatant, be inoculated with 9 after 0.22 μM of aperture filter filtering Age SPF chick embryo allantoic cavity；Be inoculated with 72 it is small when after take 50 μ L of chick embryo allantoic liquid carry out newcastle disease virus blood clotting (HA) and blood clotting suppression (HI) test.The chick embryo allantoic liquid that is positive of harvest HA and HI result of the tests, preserved after packing -70 DEG C freeze it is spare.Save To recombinant virus be named as rLa-WNV-PrM/E.

1.4：The identification of recombinant virus

1.4.1 the RT-PCR identifications of recombinant virus

In order to detect PrM/E genes whether in recombinant virus genomes, RT-PCR mirror are carried out to recombinant virus genomes It is fixed.The total serum IgE containing recombinant virus is extracted with Trizol, reverse transcription is carried out with random primer, is expanded using following primer Sense primer：WNV-PF：5 '-TGACCGACGTGATCACCATC-3 ', anti-sense primer NDV-PR：5’- CATAGGTGGTGATGAATACTGAG-3 ' is expanded, and amplification obtains the purpose fragment of 2200bp, shows recombinant viral genome Contain purpose fragment in group.

1.4.2：Indirect immunofluorescence (IFA)

RLa-WNV-PrM/E and LaSota vaccine strains respectively with MOI=0.01 infect BHK21 cells, infection 24 it is small when after Nutrient solution is discarded, with Xian PBS twice, fixes 30min with 3% paraformaldehyde room temperature afterwards, (contains 0.05%Tween-20 with PBST PBS) wash 3 times, infection cell is separately added into 1: 50 times of diluted anti-PrM monoclonal antibody of mouse (preparation of this laboratory), or E eggs White monoclonal antibody, the anti-NDV serum of chicken, room temperature effect 2h；PBST is washed three times, then is separately added into and is added 1: 200 times of diluted FITC Rabbit anti-chicken IgG (being purchased from Sigma) room temperature lucifuge of mountain sheep anti-mouse igg (being purchased from Sigma) and the TRITC mark of mark is incubated 1h, Washed 5 times with PBST, be placed under laser confocal microscope (Leica TCS SP5) and observe result.

Growth kinetics of 1.5 recombinant viruses in chicken embryo

Using rLa-WNV-PrM/E and parent plant LaSota (as control) inoculation SPF chicken embryos, respectively after inoculation 24,48, 72,96 sample when small, measure the chicken embryo median infective dose EID of each time point sample respectively₅₀。

Safety experiments of the 1.6rLa-WNV-PrM/E to mouse

By rLa-WNV-PrM/E with 1 × 10⁸EID₅₀Recombinant virus allantoic fluid intramuscular injection 0.1ml, 3 × 10⁷EID₅₀Restructuring 10 mouse are immunized in viral allantoic fluid collunarium 0.03ml, while set up LaSota vaccine strains immune group (dosage and approach and rLa- WNV-PrM/E is identical) and PBS control group.The daily set time weighs mouse weight and records health status after inoculation, continues Two weeks.

The humoral and cellular immune response evaluation of the 1.7 immune mouse of restructuring poison

Recombinant Newcastle disease virus rLa-WNV-PrM/E presses 1 × 10⁸EID₅₀It is small that 10 C57BL/6 are immunized in/an intramuscular injection Mouse, and set PBS immunized controls groups.Initial immunity after 4 weeks booster immunization, immunizing dose and approach it is identical with initial immunity.First Two weeks after secondary and booster immunization, every group takes 5 mouse to take euthanasia respectively, takes blood and separates serum, with ELISA and Neutralization test measures humoral immunity level；Mouse boosting cell is taken to be carried out with WNV E protein CD4+ and CD8+T cell epitope polypeptides Stimulate, measure total T cell shared by CD4+ the or CD8+T cells of the interferon IFN-g positives respectively with flow cytometry (FACS) Percentage, in this, as evaluation cellular immunity index.

2. result

The recombinant Newcastle disease virus structure and virus rescue of 2.1 expression WNV PrM/E albumen

Using ND LaSota vaccine strain reverse genetic operating system [19], between LaSota genome P and M genes Pme I sites insertion NDV polymerase specific recognitions gene starting and terminator sequence west nile fever virus PrM/E genes The transcriptional units independent as one, so as to be built into expression WNV PrM/E Protein reconstitution cDNA clones pBRN-FL-PrM/E. PBRN-FL-PrM/E and the helper plasmid for expressing NP, P, L albumen are transfected into BHK-21 cells jointly, 72 it is small when after harvest transfection Cell conditioned medium is simultaneously inoculated in 9 age in days SPF chicken embryos.72 it is small when after harvest the chick embryo allantoic liquid that is positive of HA and HI experiments.RT-PCR And sequencing result is shown, PrM/E genes correctly disclose into rLaSota tombs because of a group PmeI sites.Save obtained recombinant virus It is named as rLa-WNV-PrM/E.Fig. 1 shows the structure schematic diagram of the recombinant virus.

RT-PCR is using PrM/E gene specifics sense primer and NDV specific downstream primers, the results show can expand Increase the band (Fig. 2) for 2200bp or so, it was demonstrated that contain target gene PrM/E in recombinant virus genomes.

2.2PrM/E albumen can be expressed correctly in recombinant virus infection cell

PrM/E albumen can be expressed correctly in real recombinant virus as evidence, respectively with rLa-WNV-PrM/E, LaSota BHK-21 cells are infected, are carried out using PrM protein monoclonal antibodies, E protein monoclonal antibody, the anti-NDV serum of chicken as primary antibody indirect Immunofluorescence test.

The results are shown in Figure 3：The BHK-21 cells of rLa-WNV-PrM/E infection can be examined with PrM monoclonal antibodies and E protein monoclonal antibody Measure specific green florescent signal, and LaSota infected groups and be uninfected by control group and be negative, show recombinant virus rLa- WNV-PrM/E can correctly express PrM/E albumen.

E protein is to induce the most important structural proteins of WNV neutralizing antibodies and immunoprotection, therefore the correct table of E protein Up to most important to recombinant virus, in order to detect expression of the E protein in infection cell, we utilize the anti-E protein monoclonal of mouse Antibody and the anti-NDV antibody of chicken carry out immunostaining, are observed under laser confocal microscope.As a result as Fig. 4 shows that E protein exists Can be dyed in the cell of recombinant virus infection by its monoclonal antibody, show specific green florescent signal, this result shows that RLa-WNV-PrM/E can in infection cell great expression WNV E proteins.

Growth titre of 2.3 recombinant viruses in chicken embryo

RLa-WNV-PrM/E and rLa is inoculated with 9 age in days SPF chicken embryos, is sampled in different time points, measures EID₅₀, draw growth Curve.The results are shown in Figure 5, and the growth curve of rLa-WNV-PrM/E and parent's strain always, maintains the growth of high titre chicken embryo Characteristic：

Security of 2.4 recombinant viruses to mouse

By heavy dose of recombinant virus through intramuscular injection and collunarium while Mice Inoculated, pass through every daily weight of mouse after inoculation Change to reflect recombinant virus to the pathogenic of mouse.The results are shown in Figure 6, recombinant virus Mice Inoculated and parental virus and right Consistent according to group PBS immune group changes of weight trend, mouse health, does not show any clinical symptoms, it was demonstrated that rLa- in the observation period WNV-PrM/E is to mouse safety.

The humoral and cellular immune response reaction of mouse is immunized in 2.5 recombinant viruses

In order to verify the immunogenicity of recombinant virus, this research carries out immunity test to rLa-WNV-PrM/E in mouse, And the humoral immunity and cellular immune level of mouse are evaluated.The WNV PrM/E eggs of the purifying prepared using this laboratory Envelope antigen is used as in vain, quantitative analysis is carried out to the WNV specific IgG antibodies in mice serum, the results are shown in Figure 7, shows RLa-WNV-PrM/E primary immune responses can induced high levels IgG antibody, antibody level significantly rises after booster immunization：

Neutralizing antibody level most can directly react the immune protection of WNV vaccines, and E protein is induction neutralizing antibody Most important structural proteins, are almost designed currently for the vaccination of WNV both for E protein.The neutralization of this research is real Test and completed by pathogenic microorganism bio-safety National Key Laboratory of Military Medical Science Institute, experimental method is in measure flavivirus With the classical way of antibody -- plaque reduction assay, according to can suppress 50% plaque serum highest dilution be serum neutralization Potency.The results are shown in Figure 8：Recombinant virus primary immune response can produce WNV neutralizing antibodies, and neutralizing antibody is horizontal after booster immunization It is substantially increased, it is horizontal (p ＜ 0.05) is significantly higher than the neutralizing antibody after primary immune response.Equally, recombinant virus primary immune response mouse Significant E protein epitope specific T-cells immune response can be produced, t cell immune response obtains greatly adding after secondary immunity (Fig. 9) by force.In conclusion rLa-WNV-PrM/E can induce high-caliber WNV specificity neutralizing antibody and T cell is immunized, it is reason The WNV deposit vaccines thought.

After above-mentioned experimental verification, it was demonstrated that the recombinant Newcastle disease virus LaSota of expression west nile fever virus PrM/E albumen Safe and efficient West Nile fever prevention and control candidate vaccine strain is can act as, is further used for preparing the epidemic disease that can prevent West Nile fever Seedling.The vaccine strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 12nd, 2014 (CGMCC, city of BeiJing, China Chaoyang District North Star West Road 1 institute 3, Institute of Microorganism, Academia Sinica, 100101), preserving number For CGMCC No.10199.

3. discuss

West nile fever virus is a kind of important zoonosis, is distributed widely in Africa, and the European west and south is Russian, The Middle East, India and Australia.The virus incoming North America [1,2] in 1999.1999, I systems WNV epidemic diseases occurred for New York Feelings, then occurred serious epidemic situation again at 2003 and WNV in 2012 in the state, and West Nile fever in 2012 is in Italy, Israel, There are generation in Russia, Serbia, Tunisia and Greece.Birds, are the important reservoirs of WNV, and people and equus are The final host [14] of WNV.HAEMATOPHAGOUS ARTHROPODS such as mosquito etc. is the communication media of west nile virus, culex, yellow-fever mosquito, anopheles etc. Mosquito is the sick primary vehicle, has confirmed WNV in mosquito with being circulated in birds.The mankind mainly pass through viruliferous mosquito Sting and infect west nile virus.People and equus are susceptible to West Nile fever, although most of the infected's clinical symptoms are light It is micro-, but the ratio for causing nervous symptoms of the disease in recent years is higher and higher [20].20% human cases show for 3-6 days infection Show light symptoms, such as generate heat, headache, uncomfortable and fash.There are fatal nervous symptoms in 1% or so patient, such as brain ridge Marrow is scorching, encephalitis etc., and older population particularly easily sends out [7,15].The symptom of WNV infection equus (horse, donkey, mule) is similar to people, but Be 10% suffer from horse occur neurosis.Include ataxia, hind limb paralysis, muscle shake suffering from the most common nervous symptoms of Malaysia and China Quiver, atonia etc..Nervous symptoms occur suffers from the horse death rate [21-23] between 20-57%.

The present inventor utilizes newcastle disease virus LaSota vaccine strain reverse genetic operating systems, by west nile fever virus PrM/ The E genes insertion LaSota genomes transcriptional units independent as one, successfully build and save to obtain expression PrM/E albumen Recombinant Newcastle disease virus rLa-WNV-PrM/E, result of the test show that rLa-WNV-PrM/E can correctly express PrM/E albumen, and Recombinant virus maintains the high chicken embryo growth titre consistent with LaSota and the high security to mammal.

ND LaSota vaccine strain has the advantage that as carrier：Newcastle disease virus heredity is stablized relatively, and only one A serotype, occur restructuring between strain and virulence to return strong possibility minimum.NDV only transient infection on mammal, safety Property is very high；Reproduction process is completed in cytoplasm, from RNA to RNA, there is no the DNA stages and with cellular genome integrate can Energy；NDV attenuated vaccines are a kind of natural adjuvant to mammal, can induce good congenital immunity and specificity humoral and Cellular immunity is so as to produce more comprehensive, balance immunoprotection；The weak poison of NDV has the chicken embryo growth characteristics of high titre, production Cost is extremely cheap.

E protein is the most important immune protective structural proteins of WNV, and effective vaccine, which should be able to induce, to be produced in E protein And antibody.Mainly there is inactivated virus vaccine in the world currently for WNV, PrM/E protein subunit vaccines, express PrM/E eggs White DNA vaccination, and the recombinant vaccine [24- using canary pox virus or measles virus as the expression PrM/E albumen of carrier 26].In these vaccines, such as inactivated vaccine, DNA vaccination and canary pox virus vaccine merchandized handling.These vaccines Immune horse, cat, dog etc. can produce neutralizing antibody, and then produce immunoprotection [27].The expression PrM/E albumen of this research and establishment Recombinant Newcastle disease virus, primary immune response mouse can produce the WNV neutralizing antibodies of sufficient amount, neutralizing antibody water after booster immunization It is flat significantly to rise.Compareed with the international and national research work delivered at present, our vaccine can be to immune animal production Raw complete immunoprotection.Compared with other vaccines, recombinant Newcastle disease carrier bacterin is bird vaccine, therefore rLa-WNV-PrM/E Application on the birds such as the important host wild bird of WNV has inherent advantage.

Newcastle disease virus LaSota vaccine strains are a kind of outstanding live vaccine vectors, compare other kinds of vaccine (such as Asia Subunit vaccine, inactivated vaccine etc.) it can not only make body produce humoral immune reaction, its maximum advantage be can induce it is good T cell immune response, so as to provide the immunoprotection balanced for body comprehensively.We use WNV E proteins respectively in this research Specific C D4+ and CD8+T cell epitope stimulate rLa-WNV-PrM/E that the splenocyte of mouse is immunized, mouse is immunized in the results show Good immune response can be produced to this epitope.CD4+T cells are main immune auxiliary cells, they can aid in B thin Born of the same parents produce antibody, auxiliary CTL killing cells play cytotoxic effect, and CD4+T cells can produce abundant cell factor and Chemotactic factor (CF), inducing dendritic shape cell and NK cells participate in immunoreaction process.CD8+T cellular immunities mainly mediate lethal T Cell (CTL) reacts.Ctl response is rapid, can not only direct killing be infected cell, and the CTL activated can also produce including Cytokine profiles including IFN-g, so as to further enhance the immune function of body.Body is in the reset procedure of virus CTL plays key effect.Can be produced after rLa-WNV-PrM/E primary immune responses comparable levels of E protein specific C D4+ and CD8+T cells, it is ideal to illustrate that recombinant virus has effects that in terms of inducing T cell immune response.This result of study is filled Divide and confirm that rLa-WNV-PrM/E can not only make mouse produce neutralizing antibody, but also good E protein specificity can be induced T cell responses.It is contemplated that this t cell responses can play an important role in protection body is to WNV attack processes.

Although China is currently without occurring West Nile fever, the disease is as a kind of its world wide of important Amphixenosis It is widely distributed, especially waited in North America and Europe with China's trade and and export and the very close countries and regions of personnel transfer, Therefore this sick input risk is very high, its prevention and control situation allows of no optimist, and there is an urgent need to research and develop with independent intellectual property right, safety Effective West Nile fever deposit vaccine.The recombinant Newcastle disease disease of the expression west nile fever virus PrM/E albumen of this laboratory research and development Malicious LaSota vaccine strains at home and abroad belong at present to be reported first, therefore the vaccine has very strong novelty.rLa-WNV-PrM/E Good WNV neutralizing antibodies and CD4+ and CD8+T cell immune responses can be produced in mammalian animal model.rLa-WNV- PrM/E has deposit property, reply its potential threat tool of the perspective outstanding West Nile fever candidate vaccine to China as a kind of There are important strategic importance and social economic value.

It should be understood that although with reference to its exemplary embodiment, particularly shown and description is carried out to the present invention, It should be understood by those skilled in the art that without departing substantially from spirit of the invention as defined in appended claims Under conditions of scope, the change of various forms and details can be carried out wherein, can carry out any of various embodiments Combination.

Bibliography

1.Garmendia, A.E., H.J.Van Kruiningen, and R.A.French, The West Nile virus：its recent emergence in North America.Microbes and infection/Institut Pasteur, 2001.3 (3)：p.223-9.

2.Murgue, B., H.Zeller, and V.Deubel, The ecology and epidemiology of West Nile virus in Africa, Europe and Asia.Current topics in microbiology and Immunology, 2002.267：p.195-221.

3.Murray, K.O., E.Mertens, and P.Despres, West Nile virus and its Emergence in the United States of America.Veterinary research, 2010.41 (6)： p.67.

4.Brandler, S.and F.Tangy, Vaccines in development against West Nile Virus.Viruses, 2013.5 (10)：p.2384-409.

5.Austgen, L.E., et al., Experimental infection of cats and dogs with West Nile virus.Emerging infectious diseases, 2004.10 (1)：p.82-6.

6.Autorino, G.L., et al., West Nile virus epidemic in horses, Tuscany Region, Italy.Emerging infectious diseases, 2002.8 (12)：p.1372-8.

7.Sejvar, J.J., et al., Neurologic manifestations and outcome of West Nile virus infection.JAMA, 2003.290 (4)：p.511-5.

8.Petersen, L.R.and A.A.Marfin, West Nile virus：a primer for the Clinician.Annals of internal medicine, 2002.137 (3)：p.173-9.

9.Dauphin, G.and S.Zientara, West Nile virus：recent trends in diagnosis And vaccine development.Vaccine, 2007.25 (30)：p.5563-76.

10.Thakare, J.P., T.L.Rao, and V.S.Padbidri, Prevalence of West Nile virus infection in India.The Southeast Asian journal of tropical medicine and Public health, 2002.33 (4)：p.801-5.

11.Cardosa, M.J., et al., Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses.BMC Microbiology, 2002.2：p.9.

12.Oceguera, L.F., 3rd, et al., Flavivirus serology by Western blot Analysis.The American journal of tropical medicine and hygiene, 2007.77 (1)： p.159-63.

13.Koraka, P., et al., Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA.Microbes And infection/Institut Pasteur, 2002.4 (12)：p.1209-15.

14.McLean, R.G., et al., West Nile virus in livestock and Wildlife.Current topics in microbiology and immunology, 2002.267：p.271-308.

15.Mukhopadhyay, S., R.J.Kuhn, and M.G.Rossmann, A structural perspective Of the flavivirus life cycle.Nature reviews.Microbiology, 2005.3 (1)：p.13-22.

16.Pierson, T.C.and M.S.Diamond, Molecular mechanisms of antibody- mediated neutralisation of flavivirus infection.Expert reviews in molecular Medicine, 2008.10：p.e12.

17.Mehlhop, E., et al., Complement modulates pathogenesis and antibody- dependent neutralization of West Nile virus infection through a C5- Independent mechanism.Virology, 2009.393 (1)：p.11-5.

18.Fuerst, T.R., et al., Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7RNA polymerase.Proceedings of the National Academy of Sciences of the United States of America, 1986.83 (21)：p.8122-6.

19.Ge, J., et al., Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and Heterologous H5N1 avian influenza viruses.Journal of virology, 2007.81 (1)： p.150-8.

20.Petersen, L.R.and J.T.Roehrig, West Nile virus：a reemerging global Pathogen.Emerging infectious diseases, 2001.7 (4)：p.611-4.

21.Ward, M.P., et al., Investigation of an outbreak of encephalomyelitis caused by West Nile virus in 136horses.Journal ofthe American Veterinary Medical Association, 2004.225 (1)：p.84-9.

22.Cantile, C., et al., Clinical and neuropathological features of West Nile virus equine encephalomyelitis in Italy.Equine veterinary journal, 2000.32(1)：p.31-5.

23.Murgue, B., et al., West Nile outbreak in horses in southern France, 2000：The return after 35years.Emerging infectious diseases, 2001.7 (4)：p.692-6.

24.Ng, T., et al., Equine vaccine for West Nile virus.Developments in Biologicals, 2003.114：p.221-7.

25.Davis, B.S., et al., West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent Assays.Journal of virology, 2001.75 (9)：p.4040-7.

26.Minke, J.M., et al., Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus- Mosquito challenge.Archives of virology.Supplementum, 2004 (18)：p.221-30.

27.Grosenbaugh, D.A., et al., The anamnestic serologic response to vaccination with a canarypox virus-vectored recombinant West Nile virus(WNV) vaccine in horses previously vaccinated with an inactivated WNV vaccine.Veterinary therapeutics：Research in applied veterinary medicine, 2004.5(4)：p.251-7.

Claims (9)

1. the recombinant Newcastle disease virus LaSota epidemic diseases of one kind expression west nile fever virus (West Nile virus) PrM/E albumen Miao Zhu, its preserving number is CGMCC No.10199, wherein the amino acid sequence of the PrM/E albumen is as shown in SEQ ID No.2.

2. a kind of vaccine for being used to prevent the disease as caused by west nile fever virus in mammal, it includes a effective amount of right It is required that the recombinant Newcastle disease virus LaSota vaccine strains of the expression west nile fever virus PrM/E albumen described in 1.

3. vaccine according to claim 2, wherein the mammal is selected from people, horse, dog, sheep, alpaca, mouse or big Mouse.

4. vaccine according to claim 3, wherein the mammal is behaved.

5. vaccine according to claim 2, wherein the disease as caused by the west nile fever virus is selected from by West Nile fever High fever, fash, headache, aching pain of muscles, encephalitis, encephalomyelitis or relevant complication caused by virus.

6. the recombinant Newcastle disease virus LaSota vaccine strains of the expression west nile fever virus PrM/E albumen described in claim 1 exist Prepare and be used to prevent the application in mammal in the vaccine of the disease as caused by west nile fever virus.

7. application according to claim 6, wherein the mammal is selected from people, horse, dog, sheep, alpaca, mouse or big Mouse.

8. application according to claim 7, wherein the mammal is behaved.

9. application according to claim 6, wherein the disease as caused by the west nile fever virus is selected from by West Nile fever High fever, fash, headache, aching pain of muscles, encephalitis, encephalomyelitis or relevant complication caused by virus.