CN102140441A - Bird flu vaccine by using attenuated vaccinia Tian Tan as vector - Google Patents
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Abstract
The invention relates to a recombinant vaccinia virus expressing the hemagglutinin (HA) protein of H5N1 bird flu virus and the recombinant vaccinia virus can be used as the bird flu vaccine. Specifically, the bird flu vaccine is constructed on the basis of attenuated vaccinia Tian Tan (CCTCC: V200416). The invention provides a vector for constructing the bird flu vaccine. The invention also relates to an inoculation scheme by using the bird flu vaccine.
Description
Technical field
The present invention relates to biological technical field, relating in particular based on highly attenuated vaccinia virus Tiantan strain (CCTCC:V200416) is avian influenza vaccine of carrier and its production and use.
Background technology
(Avian Influenza is that the class people livestock and poultry that the A type influenza virus by orthomyxoviridae family's Influenza Virus causes suffer from communicable disease or disease syndromes altogether AI) in bird flu.1878, Perroncito reported first Italy chicken group broke out bird flu, is referred to as checken pest at that time.1901, Centannic and Sarunozzi thought that this disease is caused by " can filter " cause of disease.Up to nineteen fifty-five, just confirm that by Schafer in fact ewcastle disease virus is exactly A type avian influenza virus.Avian influenza virus now has been distributed widely in worldwide many poultry (comprising turkey, chicken, guinea fowl, quail, francolin, ostrich, pheasant, goose and duck etc.) and wild fowl (wild duck, swan, tern, aigret, guillemot, loomery and sea-gull etc.).Avian influenza virus can infected person, pig, horse, marine mammal and multiple bird, and is wherein serious to the harm of turkey and chicken.This disease is the category-A transmissible disease of OIE (OIE) regulation, and China also classifies a class zoonosis as.Discover that H hypotype and N hypotype that A type influenza virus is all are present in aquatic bird and the seabird, so aquatic bird and seabird are called as the natural storage vault of A type influenza virus.Existing studies show that, all Mammals influenza viruses all come from avian influenza virus.In 4 human flu outbreaks in 20th century (spanish influenza in 1918, nineteen fifty-seven Asia influenza, nineteen sixty-eight Mao flu and USSR (Union of Soviet Socialist Republics) influenza in 1977), the human influenza virus who is separated to all has the genetics characteristic of avian influenza virus.
According to pathogenic difference, bird flu can be divided into high pathogenic avian influenza (Highly Pathogenic Avian Influenza, HPAI), low pathogenicity bird flu (Low Pathogenic Avian Influenza Virus, LPAI) and non-virulent bird flu (None-Pathogenic Avian Influenza Virus, NPAI) three major types.The harm maximum that causes of high pathogenic avian influenza wherein.In recent years, the H5N1 high pathogenic avian influenza of frequently breaking out has brought serious economy loss not only for world's aviculture, and people's life and health also caused serious threat, Highly Pathogenic Avian Influenza Virus (HPAIV) infected person even causing death's case happens occasionally.1997, avian influenza virus direct infection people's case has appearred first in China Hong Kong Special Administrative Region, and 3 years old boy is made a definite diagnosis infects H5N1 hypotype AIV, totally 18 people's infection morbidities afterwards, 6 people's death.Thereafter, the people infects bird flu always in the fragmentary outburst in area, Asia.From in December, 2003, bird flu mainly seriously breaks out in states such as Vietnam, Thailand and Indonesia in south east asia, causes several patient deaths, and rapid spread China.At present, the many in the world countries of bird flu break out, and end on August 31st, 2009 (beginning in 2003), and The World Health Organization (WHO) reports that highly pathogenic human and bird fluenza (H5N1) confirmed cases reach 440 examples (15 countries), dead 262 examples, mortality ratio reaches 59.5%.Although also there is not evidence to show at present, the high pathogenic avian influenza H5N1 virus can be in interpersonal propagation, and the host's mutability of H5N1 virus and the characteristics of wide-scale distribution have shown should virus cause the huge possibility propagated by reorganization between the crowd.WHO thinks that this disease may be one of disease to human potential threat maximum, and the prevention and control that require countries in the world to carry out the reply flu outbreak are prepared.
Prevention may extensive popular best approach be a vaccine with the control influenza.The vaccine of flu-prevention prevailing for the time being in force mainly is divided into two kinds: with the conventional inactivated vaccine and the Gripovax of chicken embryo culture.The former is divided into whole virus, portion of product subunit SAV again.Whole virus vaccines seldom adopts because of following many side effects, and most vaccines are purified vaccine, promptly handles the virus of purifying with washing agent, is further purified hemagglutinin and neuraminic acid zymoprotein through step such as centrifugal then, prepares SAV.The antigen merging of three kinds of different virus strains also be can be made into triple vaccine.Inactivated vaccine security and immunogenicity are all good, do not exist virulence to return strong danger.But inactivated vaccine also has certain shortcoming.At first, the production of influenza virus depends on SPF chicken embryo, has limited its production in enormous quantities; Secondly, with the inactivated vaccine of chicken embryo production the part crowd is had the untoward reaction of local and whole body.In addition,, also need specific BSL-3 Biosafety production plant, limited the production of vaccine so more if be vaccine strain with highly pathogenic influenza virus.Another then is the responsive to temperature type attenuated live vaccine, and hemagglutinin gene and Neuraminidase Gene and six kinds of other responsive to temperature type attenuated strain gene recombination of the strains of influenza viruses that WHO is recommended then by the chicken embryo culture, are made attenuated live vaccine.After the attenuated live vaccine inoculation; in body, has limited fecundity; can make the reaction of body generation near inapparent infection or low-grade infection; but do not produce remarkable clinical symptom; immune effect is strong and lasting; general only the need inoculated once, and dosage of inoculation is little, has the body of stimulation and produces humoral immunization and cellular immunization protection.The shortcoming of this vaccine is exactly that validity period is short, must store under cold condition and transport, and there is hidden danger in security, and maximum shortcoming may be recovered virulence because of the producer sudden change exactly in human body.
Because the strong toxicity of H5N1, be difficult in chicken embryo growth and by height cause a disease the safety brought and regular aspect problem, limited the use ordinary method and come development and production H5N1 vaccine.In order to overcome this, develop effective H5N1 vaccine main policies at present and be because of difficulty:
1. select non-virulent strains of influenza viruses like the Antigens for use.
Since Hong Kong H5N1 in 1997 breaks out, two kinds of vaccines in the crowd, have been tried out.Use the H5N1 subunit vaccine clinical trial of non-pathogenic influenza strain A/duck/Singapore/3/97 preparation to show, though this vaccine only produces very weak antibody response through immunity repeatedly.Though use the MF59 adjuvant can improve the immunogenicity of this alternative vaccine, its protectiveness remains further to be proved (Stephenson I, et al, The Journal of Infectious Diseases, 2005 (191): 1210-1215).
2. use insect expression system to express the H5N1 surface antigen.
Use efficiently expresses proteic insect system expression H5 hemagglutinin, can protect the attack of chicken opposing H5N1 lethal dose.Clinical test results then shows very weak immunogenicity.Even through the immunity of twice 90ug high dosage, has only 52% test demonstration generation neutralizing antibody.The weak immunogenicity of the H5 hemagglutinin of insect system expression may be by due to the different glycosylations, or H5 albumen is in weak immunogenicity (Treanor JJ, et al, Vaccine, 2001 (19): 1732-1737) of human body.
3. use the reverse genetic method to make up attenuated influenza virus.
The reverse genetic ultimate principle is to use conventional influenza attenuated strain H1N1 all proteins encoded plasmids except that hemagglutinin to add the plasmid co-transfection cell through the expression H5 hemagglutinin of sudden change from the pathogenic H5N1 of height, obtains new recombinant influenza.This virus adds that by sudden change H5 albumen other components of attenuation H1N1 form.This attenuated vaccine is after the chicken embryo culture is amplified, and the influenza virus vaccine way prepares the vaccine for man of anti-H5N1 routinely.Though the influenza attenuated strain of reverse genetic transformation demonstrates effective provide protection in animal experiment.But can also have the good immune protection effect in human trial still unknown.A kind of very big may be the same with the H5 expressing protein, the weak immunogenicity of attenuated strain is difficult to produce effective neutralizing antibody at the human body internal stimulus.In addition, the reverse genetic technology is difficult to the vaccine that provides a large amount of at short notice.Therefore, present vaccine research is made in the near future at the effective anti-H5N1 vaccine of the mankind and is allowed of no optimist.Must use new research method to solve the threat of H5N1 at the extensive stream of the mankind takes place.
The live vector vaccine is that the gene with the coding viral protein inserts in other viral genome and infection animal of using or human body, produces the immune response to gene product and carrier.This have the potential immunogenicity based on the organic vaccine of reorganization, and do not have the pathologic reaction that provirus causes.Therefore become the important research field of vaccines such as HIV, SARS.Wherein with going deep into the most that the live vector vaccine of vaccinia virus vector is studied with extensive, at present successful expression the multiple gene that derives from the animal and human viroid, G protein gene as bovine epizootic fever virus, hepatitis B virus SS1 protein gene, Measles virus HA and F protein gene, rabies virus G protein and N protein gene, the GAG of acquired immune deficiency syndrome (AIDS) virus and POL protein gene etc.
In view of the difficulty that current avian influenza vaccine development exists, the present invention inserts the HA gene of H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) in the vaccinia virus Tiantan strain genome, and having prepared based on attenuated vaccinia virus Tiantan strain is the avian influenza vaccine of carrier.It has the following advantages: vaccinia virus Tiantan strain is Chinese distinctive vaccinia virus strain, Ceng Zuowei prevention smallpox popular vaccine strain used in a large amount of crowd of China midium or long term, having weak, the safe in utilization characteristics of relative virus force, is the desirable strain that development is applicable to the genetically engineered live vector vaccine that China uses; Vaccinia virus is duplicated in the endochylema of host cells infected, non-carcinogenesis; It is big to insert the foreign gene capacity, can develop combined vaccine; Can accurately express foreign protein, and offer, stimulate body to produce cellular immunization and humoral immunization, particularly can reply by inducing producing specificity CTL its processing; Avian influenza virus vaccine than deactivation is safer, and the vaccine of ratio inverse genetic technique production is more suitable for industrial production.Therefore the vaccine of different sources can in time be provided for the cross-protection research of vaccine.
Summary of the invention
One of purpose of the present invention is to overcome the deficiencies in the prior art, and a kind of bird flu attenuated vaccinia virus recombinant strain (CGMCC:3429) is provided, and the attenuated live vaccine that is prepared by this strain has good security and immunogenicity.
The invention provides the antigenic live vector avian influenza vaccine of a kind of expression AIV, it comprises vaccinia virus Tiantan strain as carrier, and the hemagglutinin gene of wherein said vaccinia virus gene group has inserted the polynucleotide of at least a coding H5N1 hypotype AIV antigen (as hemagglutinin).
In avian influenza vaccine of the present invention, the antigenic polynucleotide of described at least a coding H5N1 hypotype AIV derive from H5N1 subtype avian influenza virus Qinghai strain isolated (A/Bar-headed Goose/Qinghai/1/05).
Purpose of the present invention realizes by the following method: the clone has the HA gene that good biological is learned proterties and immune effect from the strain isolated of H5N1 subtype avian influenza virus Qinghai, AIV HA gene subclone is gone in the vaccinia virus Tiantan strain transfer vector, structure can change AIV HA gene over to recombinant plasmid that vaccinia virus recombinant the same area efficiently expresses, with this recombinant plasmid and vaccinia virus Tiantan strain reorganization, by recombinant virus is screened, purifying, acquisition can be efficient, the safety of stably express H5N1 subtype avian influenza virus HA gene, effectively vaccinia virus recombinant vaccine strain.
More specifically, the invention provides and the following:
1. express the antigenic vaccinia virus recombinant of H5N1 subtype avian influenza virus for one kind, it as carrier, has wherein inserted the antigenic gene of at least a coding H5N1 subtype avian influenza virus with attenuated vaccinia virus in the genome of described attenuated vaccinia virus.
2. according to above 1 described vaccinia virus recombinant, wherein said H5N1 subtype avian influenza virus antigen is bird flue virus H 5 N 1 subtype hemagglutinin (HA) albumen, and the proteic aminoacid sequence of preferred described HA is (referring to the attached sequence table in back) shown in SEQ ID NO:2.
3. according to above 2 described vaccinia virus recombinants, wherein insert the proteic encoding gene of described bird flue virus H 5 N 1 subtype hemagglutinin (HA) in hemagglutinin (HA) gene coding region of described vaccinia virus gene group, the nucleotide sequence of the proteic encoding gene of preferred described HA is (referring to the attached sequence table in back) shown in SEQ ID NO:1.
4. according to above 2 described vaccinia virus recombinants, the proteic gene source of described bird flue virus H 5 N 1 subtype hemagglutinin (HA) of wherein encoding is in H5N1 subtype avian influenza virus Qinghai strain isolated (A/Bar-headed Goose/Qinghai/1/05).
5. according to each described vaccinia virus recombinant among the above 1-4, wherein the described attenuated vaccinia virus as carrier is vaccinia virus Tiantan strain (CCTCC:V200416).
6. according to above 5 described vaccinia virus recombinants, its preserving number is CGMCC No.3429.
According to any one vaccinia virus recombinant among the above 1-6 in the application of preparation in the avian influenza vaccine.
8. according to above 7 described application, wherein said avian influenza vaccine is by intramuscular injection, collunarium or Orally administered.
9. avian influenza vaccine test kit, it comprises among the above 1-6 vaccinia virus recombinant of any one.
Description of drawings
The plasmid map of Fig. 1: pZCxz (Fig. 1 (A)) and pZCxz-HA (Fig. 1 (B));
The building process of Fig. 2: rVTT-HA;
Fig. 3: the fluorescence of vaccinia virus recombinant rVTT-HA identifies that (A. infects the cell of rVTT-HA; B. contrast);
Fig. 4: (A. infects the cell of rVTT-HA in the immunostaining test of vaccinia virus recombinant rVTT-HA; B. contrast);
Fig. 5: attack the poison back four different groups mouse body weight change situation (PBS represent the contrast; I.m. be expressed as intramuscular injection; I.n. be expressed as collunarium; P.o is expressed as oral);
Fig. 6: attack the poison back four different groups the mouse survival condition (PBS represent the contrast; I.m. be expressed as intramuscular injection; I.n. be expressed as collunarium; P.o is expressed as oral);
Fig. 7: the survival rate of the immunity of poison back and control group mice every day is attacked in H5N1 subtype avian influenza virus A/Bar-headed Goose/Qinghai/1/05 strain, and (PBS represents contrast; I.m. be expressed as intramuscular injection; I.n. be expressed as collunarium; P.o is expressed as oral);
Fig. 8: the survival rate of the immunity of poison back and control group mice every day is attacked in H5N1 subtype avian influenza virus A/Vietnam/1194/04 strain, and (PBS represents contrast; I.m. be expressed as intramuscular injection; I.n. be expressed as collunarium; P.o is expressed as oral).
Embodiment
Hereinafter describe reference example in detail the present invention, described embodiment only is intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying Claim.
Embodiment 1: the structure of expressing the vaccinia virus recombinant transfer vector of avian influenza virus HA gene
With H5N1 subtype avian influenza virus A/Bar-headed Goose/Qinghai/1/05 strain (Jinhua Liu#, et al.Highly pathogenic H5N1 influenza virus infection in migra-tory birds.Science, 2005, RNA 309:1206) is a template, utilization synthetic primer (primer 1:5 ' TTGGATCCATGGAGAAAATAGTGCT3 '; Primer 2: 5 ' GGCTCGAGTTAAATGCAAATTCTGC3 '; Wherein primer 1 sequence 5 ' end adds BamH I restriction enzyme site, primer 2 sequence 5 ' end adds the XhoI restriction enzyme site), carry out HA gene amplification by RT-PCR, the HA gene of amplification comprises the entire reading frame frame of HA gene.
The HA gene of amplification is gone into (Huang in the same pZCXZ shuttle vectors of cutting with BamH I and Xho I enzyme by BamH I and Xho I restriction enzyme site subclone, X., B.Lu, W.Yu, Q.Fang, L.Liu, K.Zhuang, T.Shen, H.Wang, P.Tian, L.Zhang, and Z.Chen.2009.A novel replication-competent vaccinia vector MVTT is superior to MVA for inducing high levels of neutralizing antibody via mucosal vaccination.PLoS ONE 4:e4180), this carrier contains vaccinia virus special bidirectional promoter pSYN and pH5, can efficiently express foreign gene.The influenza virus HA gene that we insert places vaccinia virus morning/late promoter pSYN downstream, is started by this promotor and expresses.Selection markers gene green fluorescent protein (GFP) is then expressed by the control of vaccinia virus morning/late promoter pH5 promotor.The foreign gene that inserts and the both sides of GFP expression casette are the encoding sequence of vaccinia virus HA gene ORF, will obtain vaccinia virus recombinant transfer plasmid carrier called after pZCXZ-HA (plasmid map is seen Fig. 1 (B)).
Embodiment 2: the structure of expressing the vaccinia virus recombinant of avian influenza virus HA gene
Inoculate six orifice plates with the chick embryo fibroblast for preparing, treat that cell 80% covers with individual layer after, the inoculation vaccinia virus Tiantan strain (CCTCC:V200416), every hole inoculum size is 0.5MOI.37 ℃ of senses are done 2 hours, outwell infection liquid, the DMEM nutrient solution rinsing cell of usefulness antibiotic-free and calf serum three times; Then according to liposome Lipofectamin
TM2000 (available from Invitrogen company) specification sheets uses the recombinant transfer plasmid pZC of liposome method with 4 μ g purifying
XZThe chick embryo fibroblast of vaccinia virus Tiantan strain has been infected in-HA transfection, and 37 ℃ of senses are done 1 hour, outwell transfection liquid, add the DMEM nutrient solution (DMEM and calf serum are all available from Invitrogen company) that contains 5% calf serum, and 37 ℃, 5%CO
2Incubator was cultivated 48 hours, treated that typical cytopathic appears in cell, gathered in the crops this cell culture multigelation three times as transfection kind poison.
Above-mentioned transfection kind poison is pressed 10
-1, 10
-2, 10
-3The chick embryo fibroblast of individual layer is covered with in extent of dilution inoculation 80%.37 ℃ of senses are done 2 hours, outwell infection liquid, and washed cell covers the DMEM nutrient agar medium that contains 1% low melting-point agarose and is cultured to the plaque appearance.Picking green fluorescence plaque is put into DMEM nutrient solution multigelation three times, carries out the recombinant virus plaque screening purifying of a new round.After so repeating to screen 6 times, promptly obtain the vaccinia virus recombinant rVTT-HA (building process is seen Fig. 2) of purifying.According to budapest treaty, this vaccinia virus recombinant rVTT-HA is deposited in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms of BeiJing, China on November 10th, 2009, No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, postcode: 100101), preserving number is CGMCC No.3429.
Get the recombinant virus rVTT-HA inoculated into chick embryo inoblast of screening purifying, 37 ℃, 5%CO
2Incubator was cultivated 48 hours, after waiting typical cytopathic to occur, put fluorescent microscope under and observed, and as seen the cell of infective virus sends green fluorescence, and the cell of not inoculating recombinant virus be can't see the green fluorescence (see figure 3).In order to detect the expression of HA, with the recombinant virus rVTT-HA inoculated into chick embryo inoblast of screening purifying, 37 ℃, 5%CO
2Incubator was cultivated 48 hours.After waiting typical cytopathic to occur, outwell nutrient solution, with DMEM cell is cleaned three times, the methanol/acetone solution pair cell that added then 1: 1 is fixed.Discard stationary liquid after five minutes, the H5 monoclonal antibody specific (available from Santa company) that adds the PBS dilution after the rinsing, incubated at room 1 hour, the anti-mouse two that adds the horseradish peroxidase-labeled of dilution in 1: 5000 after the rinsing resists (available from China fir Bioisystech Co., Ltd in Beijing) incubated at room 1 hour, adds substrate solution after the rinsing and develops the color.The result shows that HA has obtained expressing preferably and expression product has the better immunogenicity (see figure 4) in cells infected.
Embodiment 3: reorganization the Temple of Heaven vaccinia virus avian influenza vaccine is induced the effectiveness of AIV specific immunity in animal body
1. the protectiveness research of the different immunization routes of vaccine
1) vaccine produces antibody horizontal mensuration
With intramuscular injection, collunarium and oral mode 6 week SPF Balb/c mouse in age (available from Beijing Experimental Animal Center) are carried out immunity, dosage is every 3.0 * 10
6PFU (plaque forming unit, plaque forming unit), immunity twice is spaced apart 31 days altogether.Respectively at 0 day after the first immunisation, 28 days and blood sampling in 45 days, measure hemagglutination inhibition antibody (HI) level and the microneutralization antibody horizontal (http://www.oie.int/Eng/Normes/Mmanual/A_00037.htm) of anti-H5N1 virus in the serum according to the method for OIE's recommendation.Background serum (i.e. the serum that immunity was gathered in the preceding 0 day) HI and the neutralizing antibody that found that mouse are all negative.Head exempted from (first immunisation) back 28 days, and the antibody horizontal of intramuscular injection and collunarium vaccine immunity group H5 increases, and homogeneous very; And oral vaccine group antibody horizontal also has rising, but heterogeneity, it is 0 that 2 mouse HI antibody are arranged.Exempt to exempt from back 14 days in back 45 days and two at head, intramuscular injection and collunarium vaccine group H5 antibody horizontal increase, and the mean value of HI has all reached 2
7, the mean value of neutralizing antibody is respectively 623.25 and 1112.5, and oral group of HI is then at 0-2
8Between, neutralizing antibody (sees Table 1) between 0-1778.
Table 1. head exempts from the detected result of different period HI antibody in back and neutralizing antibody level
Annotate: HI represents hemagglutination inhibition antibody; NA represents microneutralization antibody
2) attack poison back mouse body weight change and survival condition
Exempt from back 22 days with H5N1 subtype avian influenza virus A/Bar-headed Goose/Qinghai/1/05 strain (Jinhua Liu mouse two
#, et al.Highly pathogenic H5N1 influenza virus infection in migra-tory birds.Science, 2005,309:1206) attack poison, attacking the toxic agent amount is 6000MLD
50(mouse medium lethal dose) from attacking poison back 3 days, claims the mouse body weight every day, finds to attack the preceding 5 days vaccine group mouse body weight in poison back and descends to some extent, and be steady relatively subsequently, the obvious (see figure 5) of control group mice weight loss.Control group mice is being attacked poison beginning death in back 4 days, and the 5th day all dead.Oral group in attacking dead 1 respectively of poison back the 4th day, 5 days, keeps stable thereafter, do not have dead (see figure 6).
2. the cross protection Journal of Sex Research of vaccine
1) homology and heterologous antibody level
With intramuscular injection and two kinds of approach of collunarium, with vaccine rVTT-HA, rVTT-S (Huang, X., B.Lu, W.Yu, Q.Fang, L.Liu, K.Zhuang, T.Shen, H.Wang, P.Tian, L.Zhang, and Z.Chen.2009.A novel replication-competent vaccinia vector MVTT is superior to MVA for inducing high levels of neutralizing antibody via mucosal vaccination.PLoS ONE 4:e4180) and the PBS contrast with 1.5 * 10
6PFU/ amount only to 6 age in week BALB/c mouse carry out twice immunity, and exempt from 2 weeks of back in two and collect mice serum, measure homology and the HI antibody of allos H5 subtype influenza virus and the titre of microneutralization antibody, find that collunarium and intramuscular injection vaccine group homology (A/Bar-headed Goose/Qinghai/1/05) HI antibody are respectively 2
6With 2
7, microneutralization antibody mean value is respectively 501.5 and 819.6, and allos (A/Vietnam/1194/04) antibody horizontal is slightly lower with respect to homologous, and HI hangs down 1 titre, between the low 1/4-2 of neutralizing antibody (seeing Table 2).
Table 2: the level of second immunisation 2 all back mouse allos and homology H5 subtype influenza antiviral antibody
Annotate: i.n represents collunarium; I.m. represent intramuscular injection; GMTs is: the abbreviation of " Geometric mean antibody titers "
2) attack poison back dead mouse situation
Two exempt from 3 weeks of back, attack poison, and it is 100MLD with dosage that A/Bar-headed Goose/Qinghai/1/05 and A/Vietnam/1194/04 (being provided by U.S. disease prevention and control center) attack poison
50(mouse medium lethal dose).A/Bar-headed Goose/Qinghai/1/05 attacks poison group, and PBS control group and rVTT-S control group are all dead, and two groups of rVTT-HA immunity are all survived, and mouse survival condition every day statistics is seen Fig. 7.After A/Vietnam/1194/04 attacked poison, PBS and rVTT-S control group were all not dead, the (see figure 8) but immune group is all survived, and protection ratio is 100%.Take to attack behind the poison the still PBS of survival and the serum of rVTT-S control group mice, measure the HI antibody of anti-A/Vietnam/1194/04, find that HI antibody is positive.
3) attacking poison back mouse body inner virus heavily separates
Respectively at brain, liver and the lung tissue of gathering mouse in immune group and the contrast group in the 3rd day, 6 days of attacking behind the poison, take by weighing weight, grind the back and add the DMEM nutrient solution that 9 times of volumes (v/w) contain 2000IU penicillin and Streptomycin sulphate (available from Huabei Pharmaceutic Co., Ltd), with abrasive material sucking-off from mortar, twice back of freeze thawing is centrifugal, 3000r/m, 5min.Draw supernatant liquor and inoculate 9 age in days SPF chicken embryos (available from Beijing Experimental Animal Center), 0.1ml/ piece.Gather allantoic fluid behind the chicken embryo death and measure the HA activity, judge the heavy separation case (seeing Table 3) of virus with this.
Table 3: attack the heavy separating resulting of the different period viruses in poison back
From viral separating resulting, rVTT-HA can effectively reduce homology and allos virus and duplicate in that mouse is intravital.Immune group mouse after homology and allos are attacked poison the 3rd day and the 6th day institute adopt in liver, lung and the cerebral tissue, be difficult to heavily be separated to virus with the chicken embryo, may be the too low reason of virus titer, and PBS and rVTT-S control group can both be separated to.
In a word; above-mentioned all experimental results show: 1. recombinant virus rVTT-HA is behind intramuscular injection, collunarium and oral three kinds of different immunization route inoculation mouse; all can produce HI antibody and neutralizing antibody by inducing mouse; but intramuscular injection is relative better with collunarium group immune effect; the antibody that produces is than homogeneous; 100% same source protection can be provided, and the antibody heterogeneity that oral route produces.Analysis-by-synthesis, collunarium can be used as the optimal selection of clinical application, and the form that vaccine can be made sprays directly sprays into nose, and is very simple and convenient.2. recombiant vaccine rVTT-HA can provide the protection of homology and heterology for mouse, and can reduce virus effectively and duplicate in that mouse is intravital.
Claims (10)
1. express the antigenic vaccinia virus recombinant of H5N1 subtype avian influenza virus for one kind, it as carrier, has wherein inserted the antigenic gene of at least a coding H5N1 subtype avian influenza virus with attenuated vaccinia virus in the genome of described attenuated vaccinia virus.
2. vaccinia virus recombinant according to claim 1, wherein said H5N1 subtype avian influenza virus antigen are bird flue virus H 5 N 1 subtype hemagglutinin (HA) albumen, and the proteic aminoacid sequence of preferred described HA is shown in SEQ ID NO:2.
3. vaccinia virus recombinant according to claim 2, wherein insert the proteic encoding gene of described bird flue virus H 5 N 1 subtype hemagglutinin (HA) in hemagglutinin (HA) gene coding region of described vaccinia virus gene group, the nucleotide sequence of the proteic encoding gene of preferred described HA is shown in SEQ ID NO:1.
4. vaccinia virus recombinant according to claim 3, the proteic encoding gene of wherein said bird flue virus H 5 N 1 subtype hemagglutinin (HA) are to be inserted in hemagglutinin (HA) gene coding region of vaccinia virus gene group by homologous recombination.
5. vaccinia virus recombinant according to claim 2, the proteic gene source of described bird flue virus H 5 N 1 subtype hemagglutinin (HA) of wherein encoding is in H5N1 subtype avian influenza virus Qinghai strain isolated (A/Bar-headed Goose/Qinghai/1/05).
6. according to each described vaccinia virus recombinant among the claim 1-5, wherein the described attenuated vaccinia virus as carrier is vaccinia virus Tiantan strain (CCTCC:V200416).
7. vaccinia virus recombinant according to claim 6, it is vaccinia virus recombinant rVTT-HA, preserving number is CGMCC:3429.
According to any one vaccinia virus recombinant among the claim 1-7 in the application of preparation in the avian influenza vaccine.
9. application according to claim 8, wherein said avian influenza vaccine is by intramuscular injection, collunarium or Orally administered.
10. avian influenza vaccine test kit, it comprises according to any one vaccinia virus recombinant among the claim 1-7.
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