CN104109655A - Preparation method of seed viruses required by H7 subtype avian influenza vaccines and diagnostic reagents - Google Patents
Preparation method of seed viruses required by H7 subtype avian influenza vaccines and diagnostic reagents Download PDFInfo
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Abstract
The invention relates to the fields of H7 subtype avian influenza vaccines and diagnostic reagents in veterinary biological products, and particularly relates to three strains of seed viruses required by H7 subtype avian influenza vaccines and diagnostic reagents, wherein the seed viruses are rH7-02, rH7-01 and rH7-06. H7 subtype highly pathogenic avian influenza has relatively strong pathogenicity on avian and human, and the vaccines constructed by applying an avian influenza reverse genetic technology have the advantages of high safety, high titer, strong matching and the like; and the prepared inactivated vaccines are safe and reliable, can produce relatively strong immunity after immunization, can effectively prevent epidemic and propagation of the H7 subtype avian influenza, reduce economic losses caused by the H7 subtype avian influenza, reduce threat to human health, have important public health significance, and have broad application prospects.
Description
Technical field
The present invention relates to the malicious preparation method of the required kind of H7 subtype avian influenza vaccine and diagnostic reagent, belong to biological technical field.
Background technology
Bird flu is a kind of poultry of being caused by avian influenza virus and the transmissible disease of wild fowl.Avian influenza virus, according to the difference of hemagglutinin (HA) and neuraminidase (NA), can be divided into 16 HA hypotypes and 9 NA hypotypes.High pathogenic avian influenza is the deadly infectious disease of the serious threat bird that caused by some H5 or H7 subtype avian influenza virus.Some Highly Pathogenic Avian Influenza Virus (HPAIV) can also cause people's infection, morbidity, even dead.
Since nineteen fifty-nine, H7 subtype avian influenza breaks out in Europe and Oceania in succession.Enter this century, H7 subtype avian influenza epidemic situation also constantly has report.For popular in China of prevention H7 subtype avian influenza virus, carry out the prevention and control tachnical storage research for this subtype avian influenza virus, be very necessary.But the domestic and international report about H7 subtype avian influenza vaccine preparation method seldom at present.This patent provides a kind of new H7 subtype avian influenza vaccine and the malicious preparation method of the required kind of diagnostic reagent.Planting poison, as " seed " of production of vaccine, is the key point of production of vaccine.Supporting diagnostic reagent is the necessary material that vaccine quality is controlled and vaccine result of use is evaluated, and plants the necessary material that poison is supporting hemagglutination test or the production of hemagglutination-inhibition test diagnostic reagent.
Summary of the invention
This patent is determined and is prepared H7 subtype avian influenza vaccine and diagnostic reagent by reverse genetic manipulation technology first.Reverse genetic manipulation technology refers to that the specific cell of transfection, produces viral a kind of technology of desired acquisition by certain or some artificial constructed plasmids.Utilize Reverse Genetics to prepare H7 subtype avian influenza vaccine and diagnostic reagent has following advantage: 1. can make avian influenza virus virulence attenuation of, improve the biological safety of production of vaccine and use; 2. improve virus titer, reduce production costs, increase economic benefit; 3. can be that transgenation changes virus by people, vaccine strain and epidemic isolates are matched on antigen.
This patent is determined and is prepared required definite HA gene order in H7 subtype avian influenza vaccine process with Reverse Genetics first.
Embodiment
Below by embodiment, further illustrate technical scheme of the present invention, but protection scope of the present invention is not limited to this.
The recombinant plasmid of six internal gene of expression PR8 virus described in embodiment is preserved by Chinese animal health and epidemiology center construction, and 293T cell is purchased from QbioGene company; Liposome Lipofectamine2000 is purchased from Invitrogen company.
Embodiment 1
The construction process of H7 subtype avian influenza virus (rH7-02), comprises the steps:
1, synthetic and clone H7 HA Gene of H 9 Subtype AIV
According to the HA gene order of appointment of the present invention (SEQ ID NO.1), adopt the method for synthetic, be inserted in two-way expression vector (can transcribe simultaneously and produce viral RNA and mRNA), make pH-02HA plasmid.
2, the clone of other genes of H7 subtype avian influenza virus
Other genes of H7 subtype avian influenza virus are used six internal gene of PR8 virus, are inserted in two-way expression vector, make pH-PB2, pH-PB1, pH-PA, pH-NP, pH-M, pH-NS plasmid.
3, the packing of H7 subtype avian influenza virus and evaluation
Express H7 HA Gene of H 9 Subtype AIV recombinant plasmid, with the recombinant expression plasmid of expressing 6 internal gene of PR8 virus, through liposome Lipofectamine2000 cotransfection 293T cell.Transfectional cell is inoculated in to 10 age in days SPF chicken embryos through allantoic cavity, every embryo 0.1mL, two embryos of each sample inoculation, put 35 ℃ and are cultured to 72h, collect allantoic fluid, by the standard that International Office of Epizootics (OIE) is recommended, carry out blood clotting (HA) and blood clotting inhibition (HI) test for identification virus.The positive allantoic fluid of HI, the 2nd generation allantoic fluid going down to posterity with chicken embryo extracts RNA, RT-PCR amplification HA and NS gene fragment checks order.RNA pcr amplification without reverse transcription.RT-PCR amplification and sequencing result show, the virus of rescue is restructuring H7 subtype avian influenza virus, without the RNA of reverse transcription with PCR without amplified band.Obtain recombinant virus called after rH7-02.
4, the required virus multiplication method of production of vaccine
With 1: 10000 times of dilution rH7-02 virus of stroke-physiological saline solution, through allantoic cavity, inoculate 10 age in days SPF chicken embryos, 0.1mL/ embryo, results are cultivated the survival chick embryo allantoic liquid after 72h, measure HA and tire, and it is 2 that viral allantoic fluid HA tires
9, and steriling test is qualified.
5, the required virus inactivating method of production of vaccine
To in the allantoic fluid of results, add formalin to final concentration 0.2%, 2-8 ℃ of deactivation 48h, shakes up once every 2h.After deactivation 48h, 10 pieces of chicken embryos of each viral sample inoculation, every embryo 0.1mL, measures HA after hatching 72h and tires, without the allantoic fluid of a blood clotting blind passage generation again.The s-generation is still judged to viral complete inactivation without blood clotting.Result virus allantoic fluid chicken embryo goes down to posterity 2 times all without producing blood clotting, and deactivation is complete.
6, the quality examination of inactivated vaccine
By the method for < < veterinary biological product rules > >, prepare rH7-02 strain oil emulsion inactivated vaccine and carry out sterility test, physical behavior, the security detection of vaccine.Indices meets the regulation of < < veterinary biological product rules > >.
7, the immuning effect test of inactivated vaccine
50 21 age in days SPF chickens are divided into 5 groups, 10 every group at random.1-4 group is the qualified rH7-02 avian influenza vaccine of intramuscular injection quality examination respectively, immunizing dose is respectively 0.05,0.1,0.3,0.5mL/ only, the 5th group is blank group, isolated rearing.Latter 2 weeks and 3 weeks of immunity, H7 subtype avian influenza virus antibody horizontal is measured in blood sampling, and measures to virus the neutralising capacity on chicken embryo, judges the immune protective effect (table 1) of vaccine.
The different immunizing doses of H7 subtype avian influenza virus oil emulsion inactivated vaccine show the test-results that affects of immune efficacy, and the vaccine immunity dosage of developing is that 0.1mL/ only just can make vaccinated flock just can obtain the protection to H7 virus in 3 weeks after immunity.From antibody generation level, the antibody horizontal of 0.3mL/ immunizing dose group chicken is apparently higher than 0.1mL group, and 0.3mL group antibody horizontal keeps stable rising.Multiple comparisons shows, 0.3 changes difference not significantly (P > 0.05) with 0.5mL group antibody titer, illustrates under similarity condition and selects 0.3mL/ dose inoculation can reach desirable epidemic prevention effect.
The immuning effect test of table 1 restructuring H7 subtype avian influenza virus oil emulsion inactivated vaccine
Note: * denominator represents test chicken number, a minute subrepresentation is separated to viral chicken embryo number.
8, H7 subtype avian influenza virus is as the method for diagnostic reagent
The enrichment procedure of H7 subtype avian influenza virus with step 4, ablation method with step 5.With this inactivation of viruses, carry out hemagglutination-inhibition test and detect the immune effect of vaccine (HI antibody titers) matching, as shown in step 7.
Embodiment 2
The construction process of H7 subtype avian influenza virus (rH7-02) as described in Example 1, difference is, H7 HA Gene of H 9 Subtype AIV sequence is SEQ ID NO.2.Obtain recombinant virus called after rH7-01.The immuning effect test result of the inactivated vaccine of preparation is as shown in table 2.
The immuning effect test of table 2 restructuring H7 subtype avian influenza virus oil emulsion inactivated vaccine
Note: * denominator represents test chicken number, a minute subrepresentation is separated to viral chicken embryo number.
Embodiment 3
The construction process of H7 subtype avian influenza virus (rH7-02) as described in Example 1, difference is, H7 HA Gene of H 9 Subtype AIV sequence is SEQ ID NO.3.Obtain recombinant virus called after rH7-06.The immuning effect test result of the inactivated vaccine of preparation is as shown in table 3.
The immuning effect test of table 3 restructuring H7 subtype avian influenza virus oil emulsion inactivated vaccine
Note: * denominator represents test chicken number, a minute subrepresentation is separated to viral chicken embryo number.
Claims (4)
1. with Reverse Genetics, artificial constructed H7 subtype avian influenza virus, as the kind poison of H7 subtype avian influenza production of vaccine.
2. artificial constructed H7 subtype avian influenza virus as claimed in claim 1, it is characterized in that, described H7 HA Gene of H 9 Subtype AIV aminoacid sequence and SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 similarity (similarity) are higher than 90%, in average every 100 amino-acid residue sites, it is identical having 90 above sites.
3. described in claim 1, plant poison, for the production of H7 subtype avian influenza deactivation epidemic disease, attenuated vaccine or supporting diagnostic reagent.
4. described in claim 2, plant poison, for the production of H7 subtype avian influenza deactivation epidemic disease, attenuated vaccine or supporting diagnostic reagent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104693291A (en) * | 2015-02-16 | 2015-06-10 | 武汉思齐源生物科技有限公司 | Application of H7 subunit antigen in animal model evaluation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104693291A (en) * | 2015-02-16 | 2015-06-10 | 武汉思齐源生物科技有限公司 | Application of H7 subunit antigen in animal model evaluation |
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