CN104830811A - NS1 gene deleted and live-attenuated vaccine candidate strain of H9N2 subtype avian influenza virus and its establishing method and application - Google Patents
NS1 gene deleted and live-attenuated vaccine candidate strain of H9N2 subtype avian influenza virus and its establishing method and application Download PDFInfo
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Abstract
The invention relates to an NS1 gene deleted and live-attenuated vaccine candidate strain rTX-NS1-128 of H9N2 subtype avian influenza virus and its establishing method and application. The NS1 gene deleted and live-attenuated vaccine candidate strain rTX-NS1-128 is characterized in that an A/chicken/Taixing/10/2010TX strain is used as a female parent to delete the length of an NS1 gene, and only 128 amino acids indicated in SEQ ID NO.15 are reserved in an NS1 protein. The NS1 gene deleted and live-attenuated vaccine candidate strain can be replicated only in a trachea, no contact transmission occurs, and safety is good. The NS1 gene deleted and live-attenuated vaccine candidate strain is available for independent use, as an effective tool to control the H9 subtype avian influenza virus, is also useful with an inactivated vaccine to allow truer immunity protection, and is able to play an important role in controlling the H9 subtype avian influenza.
Description
Technical field
The present invention relates to a kind of method of carrying out modifying structure broad spectrum H9N2 hypotype AIV attenuated live vaccine by Reverse Genetics to H9N2 subtype avian influenza virus (AIV) NS1 length protein, the AIV related generally to is the strain of BJ-94-like and Y280-like pedigree.
Background technology
H9N2 subtype avian influenza virus is worldwide popular, spreads wide, define endemic conditions disease gradually, cause huge financial loss to the aquaculture of China in China from it in 1994 after China's Mainland chicken cluster is separated.Except infection poultry, H9N2 hypotype AIV can also infect Mammals (pig and people etc.).Although H9N2 hypotype AIV infects people just cause subclinical or slight clinical symptom, but H9N2 hypotype AIV can recombinate with other subtype avian influenza virus thus produce the influenza virus of new lethal people, as H5 subtype avian influenza virus and H7N9 subtype influenza virus.Therefore, the prevention and control of H9N2 subtype avian influenza are also significant in public health.
Vaccine immunity is the effective means of prevention and control of fowl influenza.But due to the genome of AIV be that segmented RNA viruses easily reprovision occurs, the poor replication fidelity that RNA polymerase copies easily cause virus quick variation, therefore, existing inactivated vaccine is unsatisfactory to safety control of bird flu effect.The inactivated virus vaccine that current China uses is existing for early stage strain, as Ck/SH/F/98 and Ck/SD/6/96 (BJ/94 pedigree) (Li C, Yu K, Tian G, et al Evolution of H9N2influenza viruses from domestic poultry in Mainland China.Virology, 2005,340:70-83).Also have for recent epidemic strain (Y280 pedigree) (Zhang Y, Yin Y, Bi Y, et al.Molecular and antigeniccharacterization of H9N2avian influenza virus isolates from chicken flocks between 1998and 2007in China.VetMicrobiol 2012,156,285-293.), vaccinated flock still has the Epidemic outbreak of disease of H9N2 hypotype AI frequently.Under laboratory condition, after SPF chicken group inoculates inactivated vaccine prepared by early stage strain, the H9N2 hypotype AIV of current popular is used to carry out attacking poison in collunarium mode, still can there is high-caliber toxin expelling (Sun Y, Pu J, Jiang Z, et al.Genotypic evolution and antigenic drift of H9N2influenza viruses in China from1994to 2008.Vet Microbiol, 2010,146:215-225.); And the inactivated vaccine using part epidemic strain to prepare can not protect SPF chicken to resist the attack (Liu Dong of H9N2 virus well, Gong Xiao, Liu Xiaodong, waits the screening of .2013.H9N2 subtype avian influenza Epidemic analysis and vaccine strain. Chinese Animal Quarantine .2013.30. (6): 45-50; Wan Xiaopeng, Zeng Xianying, Tian Guobin, wait the screening of .H9N2 subtype avian influenza epidemic strain inactivated vaccine kind poison. and Chinese Preventive Veterinary Medicine reports .2010.32 (4): 277-280).Therefore, the vaccine that there is intersecting protective for different pedigree H9 hypotype AIV strain is developed extremely urgent.
Compared with traditional inactivated vaccine, attenuated live vaccine can induce body to produce mucous membrane and cellullar immunologic response, has the advantage that cross-protection is good.And attenuated live vaccine is immune, easy to use by the mode of spraying or drinking-water, greatly reduces human cost.For live Newcastle disease vaccine, do not need inject and only just can complete mass immunization (the Senne DA of chicken quickly and easily with drinking water, King D J, KapczynskiD R.Control of Newcastle disease by vaccination.Developments in biologicals, 2004,119165-70.).Some researchs show, can attenuated virus (Quinlivan M by the activity of the NS1 albumen hindering or reduce influenza virus, Zamarin D, Garcia-Sastre A, etal.Attenuation of equine influenza viruses through truncations of the NS1protein.Journal of virology, 2005, 79 (13): 8431-9.Zhou B, Li Y, Belser JA., et al.NS-based live attenuated H1N1pandemic vaccines protect mice andferrets.Vaccine, 2010, 28 (50): 8015-8025.), and NS1 albumen is Nonstructural Protein, it is carried out transforming the antigenicity that can't affect virus itself.Along with development and the maturation of influenza virus Reverse Genetics, can modify the virulence associated gene of virus in vitro and obtain fast and cause weak recombinant virus, obtain potential attenuated live vaccine Candidate Strain.In this research, we are with A/chicken/Taixing/10/2010 (TX) strain (Zhu Y, Yang, Liu W, et al.Comparison of biological characteristics of H9N2avian influenza viruses isolatedfrom different hosts, Archives of virology, 2015, be 160:917-927) maternal, to the disappearance that the length of its NS1 gene is carried out in various degree, NS1 albumen is made to retain 73 respectively, 100 and 128 amino acid, 3 strain H9 hypotype AIV NS1 gene-deleted strains are saved out by Reverse Genetics.Measure the virulence of gene-deleted strain and immune efficacy, pick out virulence minimum and the recombinant virus of immune efficacy can be kept as vaccine candidate strain simultaneously, observe it after immune chicken and the protection cross-protection that poison provides is attacked to different pedigree H9 hypotype AIV.
Summary of the invention
H9N2 hypotype AIV infects and causes huge financial loss to aviculture, and the main H9N2 subtype avian influenza inactivated vaccine that uses carrys out this disease of prevention and control at present.Due to the HI antibody that H9N2 subtype avian influenza inactivated vaccine can only induce generation higher, can not provide cellular immunization and mucosa-immune, the H9N2 hypotype AIV of commute generation antigenic variation is difficult to provide cross protection, usually immuning failure occurs.Therefore, be necessary to develop energy inducing cell, body fluid and mucosa-immune, produce the novel attenuated live vaccines of intersecting protective.
Be maternal with A/chicken/Taixing/10/2010 (TX) strain of the Y280 pedigree of current popular, to the disappearance that the length of NS1 gene is carried out in various degree, make NS1 albumen retain 73,100 and 128 amino acid respectively; The aminoacid sequence of wild-type NS1 albumen is as shown in SEQ IDNO.12; Above-mentioned reservation 73,100 and 128 amino acid whose sequences are SEQ ID NO.13, SEQ ID NO.14 and SEQ IDNO.15 respectively.Reverse Genetics is utilized to save out 3 strain H9 hypotype restructuring AIV.Measure Virulence Indices and the immune antibody production of recombinant virus; filter out the good and recombinant virus that immunogenicity is good of security as vaccine candidate strain; immunity chicken postevaluation its provide protection that poison provides is attacked to different pedigree H9 hypotype AIV, filter out the attenuated live vaccines that cross protection can be provided.
The technical solution adopted in the present invention is: H9N2 subtype avian influenza virus NS1 gene-deletion attenuated living vaccine Candidate Strain rTX-NS1-128, it is for female parent with A/chicken/Taixing/10/2010TX strain, the length of NS1 gene is lacked, makes NS1 albumen only remain 128 amino acid; 128 described amino acid whose sequences are as shown in SEQ ID NO.15.
The present invention further discloses the construction process of rTX-NS1-128, is with A/chicken/Taixing/10/2010TX strain for female parent, with NS full length gene primer NS-1 and NS-2 reverse transcription PCR amplification NS gene, is cloned into
carrier, constructs positive plasmid pCR2.1-TX-NS, is template, combinationally uses primer NS-1 and NS-128-OL-F with pCR2.1-TX-NS, and NS-128-OL-R and NS-2 amplifies NS1 gene epimere and hypomere band respectively; After 1:1 mixing, primer NS-1 and NS-is used to amplify the NS-128 full-length gene of 810bp as template two sections; By this gene clone extremely
carrier, builds positive plasmid pCR2.1-TX-NS1-128; Carry out being connected after pCR2.1-TX-NS1-128 is cut with Bsm I enzyme respectively with transcriptional expression plasmid pHW2000 and build transcriptional expression plasmid pHW2000-TX-NS1-128; By the host cell of transcriptional expression plasmid co-transfection avian influenza virus license obtained, results Transfected cells supernatant liquor inoculated into chick embryo allantoic cavity is rescued recombinant virus rTX-NS1-128.
The invention also discloses described H9N2 subtype avian influenza virus NS1 gene-deletion attenuated living vaccine Candidate Strain rTX-NS1-128 and prepare the application in broad spectrum H9N2 hypotype AIV attenuated live vaccine.
This vaccine candidate strain only can copy in tracheae, does not come in contact propagation, and security is good.Higher HI antibody can be produced after immunity chicken, good cross protection can be provided to the H9 hypotype AIV in popular two the pedigree BJ/94 and Y280 of China.This vaccine candidate strain both can be used alone, and became the effective tool of H9 subtype avian influenza prevention and control, also can with inactivated vaccine conbined usage, more certain immanoprotection action is provided.Very important effect is played by the prevention and control of H9 subtype avian influenza.
Accompanying drawing explanation
Fig. 1 is that NS1 genetically deficient recombinant virus builds route map.
Fig. 2 is PCR primer (the M:100bp mark of NS1 genetically deficient; 1:WT-TX; 2:rTX-NS1-73; 3:rTX-NS1-100; 4:rTX-NS1-128).
Fig. 3 is that H9 parental virus TX and rTX-NS1 genetically deficient restructuring poison copy situation in chicken tracheal tissue.
Fig. 4 is that H9 parental virus TX and rTX-NS1 genetically deficient restructuring poison copy situation in chicken lung tissue.
Fig. 5 is the antibody time length monitoring of recombinant virus rTX-NS1-128 immunity chicken induction.
Embodiment
Testing program concrete in the present invention:
1. the rescue of genetically deficient and vaccine strain
1.1 design of primers
According to A/chicken/Taixing/10/2010 (TX) strain virus NS gene order (Genbank sequence number: JN653684, A/chicken/Taixing/10/2010 (TX) strain (Zhu Y, Yang Y, Liu W, et al.Comparison of biological characteristics ofH9N2avian influenza viruses isolated from different hosts, Archives of virology, 2015,160:917-927) design NS1 genetically deficient primer (table 1).
Table 1 design of primers
1.2Overlapping-PCR carries out disappearance to NS1 gene and modifies
With TX strain totivirus nucleic acid for template, total length primer (NS-1 and NS-2) reverse transcription PCR (RT-PCR) increases NS gene, adopts 25 μ L systems: 10 × Buffer 2.5 μ L, Mg
2+the each 0.5 μ L of 1.5 μ L, dNTP 0.5 μ L, primer NS-1 and NS-2 (concentration is 25 μm of ol/ μ L), Taq DNA polymerase (1U/ μ L) 0.5 μ L, DNA profiling 2.5 μ L, mend to 25 μ L with aseptic ultrapure water.Response procedures: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45s, 54 DEG C of annealing 45s, 72 DEG C extend 60s, 30 circulations, and 72 DEG C extend 10min.Be added to after PCR primer being added sample-loading buffer in the sepharose of 1%, with 100bp marker for standard reference, with 80V electrophoresis 1h, observations after dyeing, TX strain NS gene fragment total length is 879bp.Gel-purified reclaims test kit and reclaims PCR primer, measures concentration, is cloned into
vector (purchased from Invitrogen company), connect product conversion competent cell, choose spot, little upgrading grain, plasmid send Jin Sirui biotechnology company limited sequence verification, positive plasmid called after pCR2.1-TX-NS.
Take pCR2.1-TX-NS as template, use total length primer (NS1 and NS-73-OL-R) and (NS-73-OL-F and NS-2) respectively pcr amplification go out NS gene upper (233bp), under (437bp) two sections, two sections are amplified full-length gene SEQ ID NO.9 (645bp) of NS-73 (NS1 albumen retains 73 amino acid) as template use primer (NS1 and NS2) after 1:1 mixing.Take pCR2.1-TX-NS as template, use total length primer (NS1 and NS-100-OL-R) and (NS-100-OL-F and NS-2) respectively pcr amplification go out NS gene upper (314bp), under (437bp) two sections, two sections are amplified full-length gene SEQ ID NO.10 (726bp) of NS-100 (NS1 albumen retains 100 amino acid) as template use primer (NS1 and NS2) after 1:1 mixing.Take pCR2.1-TX-NS as template, use total length primer (NS1 and NS-128-OL-R) and (NS-128-OL-F and NS-2) respectively pcr amplification go out NS gene upper (398bp), under (437bp) two sections, two sections are amplified full-length gene SEQ ID NO.11 (810bp) (Fig. 2) of NS-128 (NS1 albumen retains 128 amino acid) as template use primer (NS1 and NS2) after 1:1 mixing.Gel-purified reclaims test kit and reclaims PCR primer, measures concentration, is cloned into
vector, connects product conversion competent cell, chooses spot, little upgrading grain, plasmid send Jin Sirui biotechnology company limited sequence verification, and positive plasmid is called after pCR2.1-TX-NS1-73, pCR2.1-TX-NS1-100 and pCR2.1-TX-NS1-128 respectively, saves backup in-20 DEG C.
1.3 expression plasmids build
Positive plasmid (pCR2.1-TX-NS1-73, pCR2.1-TX-NS1-100 and pCR2.1-TX-NS1-128) cut with restriction enzyme Bsm I (NEB Products) enzyme, obtain NS gene fragment, by itself and the pHW2000 (HoffmannE cut through Bsm I (NEB Products) enzyme, Neumann G, Kawaoka Y, et al.A DNA transfection system for generation of influenza A virus from eightplasmids.Proc.Natl.Acad.Sci.U.S.A.2000, 97, 6108-6113.) connect, transform, picking positive plasmid is identified.Positive plasmid is called after pHW2000-TX-NS1-73 respectively, pHW2000-TX-NS1-100 and pHW2000-TX-NS1-128.Plasmid kit (QIAGEN Products) extracting is proposed with little by after the expression plasmid enlarged culturing of acquisition, the transcriptional expression plasmid (pHW2000-TX-PB2, pHW2000-TX-PB1, pHW2000-TX-PA, pHW2000-TX-HA, pHW2000-TX-NP, pHW2000-TX-NA, pHW2000-TX-M) simultaneously preparing other 7 genes of TX strain (builds voluntarily, method is with NS gene), measure plasmid concentration respectively, and its concentration is all adjusted to 300ng/ μ L and is used for transfection.
The rescue of 1.4 viruses
Virus rescue is with reference to (Hoffmann E such as Hoffmann, Neumann G, Kawaoka Y, et al.A DNA transfection system forgeneration of influenza A virus from eight plasmids.Proc.Natl.Acad.Sci.U.S.A.2000,97,6108-6113.) method carries out.Respectively the transcriptional expression plasmid of 3 NS1 genetically deficients is joined in 1.5ml dactylethrae with other 7 each 300ng of plasmid of TX and mix.In dactylethrae, add 100 μ L again do not contain in the DMEM substratum of microbiotic and serum, micropipet is blown and beaten plasmid is fully mixed in DMEM substratum gently, adds 10 μ L transfection reagents and again mixes, in room temperature, act on 20min.MDCK and the 293T cell (1:2, degrees of fusion is at about 60-80%) of the Dual culture in 6mm dish is washed 2 times by nonreactive without blood DMEM, then adds the DMEM that 1ml contains 10% foetal calf serum.After liposome effect completely, the DMEM of 10% of 800 μ L is added with termination reaction in dactylethrae, afterwards the liquid in dactylethrae is dropwise joined in culture dish.By culture dish after 37 DEG C of CO2gas incubator cultivate 8-10h, adding TPCK pancreatin to final concentration is 2 μ g/ml, after about 60h is cultivated in 37 DEG C of continuation, is sealed by whole culture dish, be placed in-70 DEG C of refrigerator multigelations three times with sealed membrane.Collect and inoculate 7 age in days SPF chicken embryos (0.3ml/ embryo) through the cell conditioned medium of freeze thawing treatment, cultivate in 35 DEG C of incubators.After cultivating 72h, the HA surveying chick embryo allantoic liquid tires, and collects the highest allantoic fluid of tiring.Then, the allantoic fluid of results being inoculated instar chicken embryo on the 10th and carry out continuous passage, passed for 5 generations continuously, by the recombinant virus of rescue called after rTX-NS1-73, rTX-NS1-100 and rTX-NS1-128 respectively, and preserving being stored in-70 DEG C of Ultralow Temperature Freezers after its packing.Measure the chicken embryo median infective dose (EID of recombinant virus
50), result display recombinant virus rTX-NS1-73, the EID of rTX-NS1-100 and rTX-NS1-128
50be respectively 7.47,7.66 and 7.6Log
10eID
50/ 0.1ml, than the value (8.08Log of parental virus
10eID
50/ 0.1ml) lower slightly.
2.SPF chicken pathogenic is tested
In order to measure pathogenic to chicken of recombinant virus that parental virus and NS1 gene lack in various degree.44 4 week age SPF chicken, random packet, often organize 11, with 10
6eID
50/ 200 μ L/ virus quantity only attacks poison in collunarium mode, attacks the rear Continuous Observation of poison 21 days.
2.1 recombinant viruses organize replication to measure
In order to measure the replication of recombinant virus in chicken organ-tissue, within the 3rd day after attacking poison and the 5th day, slaughtering 3 chickens respectively, collecting respiratory tract and lung tissue.Get the tissue that a part is collected, electronic homogenised tissue after adding PBS in the ratio of 1g/0.3ml, 8,000rpm × 10min collected after centrifugation supernatant.Supernatant is inoculated SPF chicken embryo and measure EID
50with the virus titer in titration tracheae and lung tissue.All recombinant viruses all can not cause obvious clinical symptom after infected chicken.Virus is content titrimetry display in tracheal tissue, after virus inoculation the 3rd day, the highest (5.00 ± 0.43log of titre of parental virus TX group
10eID
50/ g), the titre of rTX-NS1-73 group is taken second place (3.75 ± 0.66log
10eID
50/ g), rTX-NS1-100 group third (3.66 ± 0.80log
10eID
50/ g), the minimum (3.16 ± 0.64log of titre of rTX-NS1-128 group
10eID
50/ g); After virus inoculation the 5th day, only has parental virus TX (2.83 ± 0.29log
10eID
50/ g) and recombinant virus rTX-NS1-73 (1.74 ± 0.20log
10eID
50/ g) can copy (Fig. 3).Statistical analysis in viral tracheal tissue is shown, after virus inoculation the 3rd day, only have the titre of rTX-NS1-128 group significantly lower than parental virus TX group (P<0.05).Virus is content titrimetry display in lung tissue, after virus inoculation the 3rd day, only has parental virus TX to copy; After virus inoculation the 5th day, parental virus TX also cannot copy (Fig. 4).
2.2 recombinant virus toxin expellings measure
In order to detect the toxin expelling situation of recombinant virus in chicken.After attacking poison the 3rd day, the 5th day and within the 7th day, gather respiratory tract and the cloacal swabs of chicken respectively, after process, inoculated into chick embryo detection toxin expelling situation.Result display the 3rd day after inoculation, the toxin expelling rate the highest (100%) of parental virus TX group, and the toxin expelling rate of rTX-NS1-128 group minimum (80%), and between parental virus group, difference is not remarkable.After virus inoculation the 5th day, parental virus TX group still keeps the toxin expelling rate of 100%, the toxin expelling rate of rTX-NS1-73 and rTX-NS1-100 group slightly declines, the toxin expelling rate of rTX-NS1-128 group then drops to 50%, significantly lower than the toxin expelling rate (p<0.05) of parental virus group.After virus inoculation the 7th day, parental virus TX group is only had still to present low-level toxin expelling (20%), recombinant virus group all not toxin expellings.Carry out qualitative analysis to cloacal swabs, result shows, and only has parental virus TX group can by cloaca high-level efficiency toxin expelling, and 3 strain NS1 genetically deficient recombinant virus groups are not all by cloaca toxin expelling (table 2).
Table 2.NS1 genetically deficient is recombinated malicious toxin expelling situation
alarynx tracheal swab;
bcloacal swab
Quantitative analysis is carried out to being defined as positive cotton swab through qualitative detection.Result show, rTX-NS1-73, rTX-NS1-100 and rTX-NS1-128 group after virus infection the 3rd day the virus titer gathered in tracheal swab be respectively 3.55 ± 0.26log
10eID
50/ 0.1ml, 2.94 ± 0.50log
10eID
50/ 0.1ml and 1.94 ± 0.27log
10eID
50/ 0.1ml.And the virus titer of parental virus TX group is 6.05 ± 0.55log
10eID
50/ ml, is significantly higher than (P<0.05) arbitrary recombinant virus group.Statistical analysis discovery is carried out to recombinant virus titre, significant difference (P<0.05) between rTX-NS1-128 and rTX-NS1-73 group, and and between rTX-NS1-100 group, difference is not significantly (table 3).
The quantitative analysis of table 3. viral tracheal swab toxin expelling
2.3 recombinant virus immunogenicity determinings
Infect latter 21 days, gather chicken serum, detect the HI antibody titer in serum, judge its humoral immunoresponse(HI) level.Result shows, and NS1 genetically deficient recombinant virus group is the same with parental virus group, and body can be induced to produce desirable immunne response level, and its average HI tires all between 9-10log2 (table 2).
In sum, recombinant virus rTX-NS1-128 is compared with recombinant virus rTX-NS1-73, rTX-NS1-100 and parental virus TX, virulence is the most weak, security is the highest, and still keep good immunogenicity, therefore select recombinant virus rTX-NS1-128 to do further security and immune efficacy evaluation.
3. the propagated test of chicken and antibody continue duration monitoring
The propagation test of 3.1 viruses in chicken
50 SPF chickens, random packet, wherein often organizes 5 and inoculates different extent of dilution respectively (from 10 by 10/group
3-10
7eID
50totally 5 extent of dilution) virus as donor group, after it attacks malicious 24h, in same cage, put into 5 chickens as contact group.Attack poison and the 3rd, 5 after exposing with within 7 days, to gather respectively attack malicious group with the tracheae and the cloacal swab that contact chicken in group, after process, inoculated into chick embryo measures toxin expelling situation.After infecting or expose 21 days, blood sampling, measures its HI antibody titer, observes serum simultaneously and turns positive phenomenon.Result shows, when parental virus is respectively with 10
5eID
50, 10
6eID
50with 10
7eID
50after dose inoculation, the chicken of all inoculation group is all by respiratory tract and cloaca high-level efficiency toxin expelling; Chicken in contact group after exposure the 3rd day and the 5th day also by respiratory tract and cloaca toxin expelling, and toxin expelling level and toxin expelling time with attack poison and organize similar.And when dosage of inoculation is 10
4eID
50, inoculation group declines all to some extent with the toxin expelling efficiency of the chicken contacted in group.When dosage of inoculation is 10
3eID
50time, although fail to be separated to virus from inoculation group and the cotton swab contacting group chicken, all chickens are in inoculation or expose latter 21 days and all there occurs strong serum and turn positive phenomenon, demonstrate high contact transmission ability (table 4).
Different from parental virus, recombinant virus rTX-NS1-128 has completely lost contact transmission ability, even if use high to 10
7eID
50the virus of dosage is inoculated, and the chicken of all contact groups is not all propagated by respiratory tract and cloaca, and any serum does not occur after exposure on the 21st day turns sun.And the chicken of direct infection all occurs that serum turns sun after inoculation for 21 days.And use 10
5eID
50when dosage is tested, although all do not isolate virus in the tracheae of chicken and cloaca from inoculation group, inoculating latter 21 days still has part chicken generation serum to turn positive phenomenon (table 5).
Table 4. parental virus dose response and propagation test
ainoculation or exposure, after 21 days, gather chicken serum and detect HI valency.If HI is value <4log2, then judge that serum does not occur turns positive phenomenon
Table 5. recombinant virus dose response and propagation test
ainoculation or exposure, after 21 days, gather chicken serum detection HI and tire.If HI<4, judge that serum does not occur turns positive phenomenon
3.2 antibody continue duration monitoring
In order to observe antibody horizontal and time length, by the recombinant virus rTX-NS1-128 of NS1 genetically deficient that filters out with 10
6eID
50after the dosage of/200 μ L inoculates SPF chicken in 4 week age in collunarium mode, collected serum every 7 days and measure its HI and tire.As shown in Figure 5, after immunity, the 2nd week antibody horizontal significantly rises antibody test result, reaches peak value (10log to the 3rd week HI
2left and right), high-caliber HI valency continues about 8 weeks afterwards.After 9th week, antibody horizontal starts to decline, and drops to 5log to the 11st week HI antibody titers
2left and right.
4. Immunoprotection test
40 4 week age SPF chicken, random packet, immune group is with collunarium mode immunity 10
6eID
50the rTX-NS1-128 of/200 μ L, establishes PBS group in contrast simultaneously.After immune 21 days, with 10
6eID
50homotype (homology virus TX and the heterologus virus F98) virus of/200 μ L dosage attacks poison by collunarium mode.Within after attacking poison the 3rd, 5 and 7 day, gather tracheae and cloacal swabs respectively, process cotton swab, inoculation SPF chicken embryo, isolated viral, detects toxin expelling situation.
Protest test result shows; no matter chicken through rTX-NS1-128 immunity accepts the attack of homology (TX strain) or allos (F98 strain) H9N2 subtype avian influenza virus; after attacking poison, all do not isolate virus in the tracheae gathered of the 3rd, 5 and 7 day and cloacal swabs, toxin expelling rate is 0%.And control group is attacking poison latter 3rd day and the 5th day, all by respiratory tract and the efficient toxin expelling of cloaca, toxin expelling rate, up to more than 90%, is being significantly higher than (p<0.05) arbitrary immune group (table 6).
Protest test after table 6.SPF chicken immune
atracheal swab;
bcloacal swab.
Claims (4)
1.H9N2 subtype avian influenza virus NS1 gene-deletion attenuated living vaccine Candidate Strain rTX-NS1-128, it is characterized in that, it be with A/chicken/Taixing/10/2010 TX strain for female parent, the length of NS1 gene is lacked, makes NS1 albumen only remain 128 amino acid; 128 described amino acid whose sequences are as shown in SEQ ID NO.15.
2. H9N2 subtype avian influenza virus NS1 according to claim 1 gene-deletion attenuated living vaccine Candidate Strain rTX-NS1-128, it is characterized in that being obtained by the method for giving: be for female parent with A/chicken/Taixing/10/2010 TX strain, with NS full length gene primer NS-1 and NS-2 reverse transcription PCR amplification NS gene, be cloned into
carrier, constructs positive plasmid pCR2.1-TX-NS, is template, combinationally uses primer NS-1 and NS-128-OL-F with pCR2.1-TX-NS, and NS-128-OL-R and NS-2 amplifies NS1 gene epimere and hypomere band respectively; After 1:1 mixing, primer NS-1 and NS-is used to amplify the NS-128 full-length gene of 810bp as template two sections; By this gene clone extremely
carrier, builds positive plasmid pCR2.1-TX-NS1-128; Carry out being connected after pCR2.1-TX-NS1-128 is cut with Bsm I enzyme respectively with transcriptional expression plasmid pHW2000 and build transcriptional expression plasmid pHW2000-TX-NS1-128; By the host cell of transcriptional expression plasmid co-transfection avian influenza virus license obtained, results Transfected cells supernatant liquor inoculated into chick embryo allantoic cavity is rescued recombinant virus rTX-NS1-128.
3. the construction process of H9N2 subtype avian influenza virus NS1 according to claim 1 gene-deletion attenuated living vaccine Candidate Strain rTX-NS1-128, it is characterized in that, for female parent with A/chicken/Taixing/10/2010 TX strain, with NS full length gene primer NS-1 and NS-2 reverse transcription PCR amplification NS gene, be cloned into
carrier, constructs positive plasmid pCR2.1-TX-NS, is template, combinationally uses primer NS-1 and NS-128-OL-F with pCR2.1-TX-NS, and NS-128-OL-R and NS-2 amplifies NS1 gene epimere and hypomere band respectively; After 1:1 mixing, primer NS-1 and NS-is used to amplify the NS-128 full-length gene of 810bp as template two sections; By this gene clone extremely
carrier, builds positive plasmid pCR2.1-TX-NS1-128; Carry out being connected after pCR2.1-TX-NS1-128 is cut with Bsm I enzyme respectively with transcriptional expression plasmid pHW2000 and build transcriptional expression plasmid pHW2000-TX-NS1-128; By the host cell of transcriptional expression plasmid co-transfection avian influenza virus license obtained, results Transfected cells supernatant liquor inoculated into chick embryo allantoic cavity is rescued recombinant virus rTX-NS1-128.
4. H9N2 subtype avian influenza virus NS1 according to claim 1 gene-deletion attenuated living vaccine Candidate Strain rTX-NS1-128 is preparing the application in broad spectrum H9N2 hypotype AIV attenuated live vaccine.
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| CN108220217A (en) * | 2018-01-25 | 2018-06-29 | 上海理工大学 | A kind of attenuation Listeria Monocytogenes and its application for being used to delivering and expressing exogenous antigen |
| US10098945B2 (en) | 2004-06-01 | 2018-10-16 | Icahn School Of Medicine At Mount Sinai | Genetically engineered swine influenza virus and uses thereof |
| CN109880835A (en) * | 2019-03-28 | 2019-06-14 | 扬州大学 | Recombinate H9N2 avian flu strain, preparation method, avian influenza vaccine and its application |
| CN111647610A (en) * | 2020-06-02 | 2020-09-11 | 扬州大学 | H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals and construction method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10098945B2 (en) | 2004-06-01 | 2018-10-16 | Icahn School Of Medicine At Mount Sinai | Genetically engineered swine influenza virus and uses thereof |
| US10543268B2 (en) | 2004-06-01 | 2020-01-28 | Icahn School Of Medicine At Mount Sinai | Genetically engineered swine influenza virus and uses thereof |
| CN108220217A (en) * | 2018-01-25 | 2018-06-29 | 上海理工大学 | A kind of attenuation Listeria Monocytogenes and its application for being used to delivering and expressing exogenous antigen |
| CN108220217B (en) * | 2018-01-25 | 2021-02-09 | 上海理工大学 | Attenuated listeria monocytogenes for delivering and expressing exogenous antigen and application thereof |
| CN109880835A (en) * | 2019-03-28 | 2019-06-14 | 扬州大学 | Recombinate H9N2 avian flu strain, preparation method, avian influenza vaccine and its application |
| CN109880835B (en) * | 2019-03-28 | 2022-03-25 | 扬州大学 | Recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof |
| CN111647610A (en) * | 2020-06-02 | 2020-09-11 | 扬州大学 | H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals and construction method and application thereof |
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