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Preparation method and application of virus Ankara subunit vaccine

An Ankara virus and subunit vaccine technology, applied in the field of Ankara virus subunit vaccine preparation, can solve problems such as toxic organisms, adverse immune responses, economic losses and the like

Inactive Publication Date: 2017-05-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease has caused such a large economic loss that it is urgent to put a safe and efficient vaccine into the market
However, the commonly used inactivated vaccines may damage or change effective antigenic determinants during the inactivation process; the immune effect produced is short-lived and does not produce local antibodies; it may produce toxicity or potential adverse immune responses to the body; One injection requires a relatively large amount of antigen, and the cost is relatively high

Method used

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  • Preparation method and application of virus Ankara subunit vaccine
  • Preparation method and application of virus Ankara subunit vaccine
  • Preparation method and application of virus Ankara subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Embodiment 1: Acquisition of recombinant baculovirus Ac-Penton

[0106]1. Acquisition of Penton gene

[0107] Extraction of Ankara virus DNA:

[0108] Viral DNA was extracted using the SDS-Protease K-phenol / chloroform method, that is, 600 μL of the virus liquid was centrifuged at 4°C and 12,000 rpm / min for 10 minutes, and 425 μL of the supernatant was added to 20 μL of 25 mg / mL proteinase K and 50 μL of 10% SDS to make it Proteinase K and SDS final concentrations were 1 mg / ml and 1%. Water bath at 56°C for 1 hour and shake occasionally; add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and mix thoroughly, centrifuge at 10,000 rpm at 4°C for 15 minutes. Take the supernatant, add an equal volume of chloroform to the supernatant and mix thoroughly at 4°C at 10,000 rpm, centrifuge for 15 minutes, take the supernatant, add 1 / 10 volume of NaAc (3M, pH 5.0) and more than 2 times the volume of absolute ethanol and mix well. Precipitate at -20°C for 2 hours, ...

Embodiment 2

[0148] Embodiment 2: Expression, quantification and purification of recombinant protein Penton

[0149] 1. Western Blotting to detect the expression of the target protein Penton

[0150] Cells infected with P3 recombinant baculovirus and normal cells were lysed with cell lysate on ice for 30 minutes, added to 5×SDS Loading Buffer, boiled in boiling water for 5-8 minutes, and then separated, followed by conventional methods for 10 minutes. SDS-PAGE electrophoresis analysis of % separating gel and 5% stacking gel, transfer to PVDF membrane with 5% skimmed milk powder, block overnight at 4°C, add 1:5000 diluted His tag primary antibody, shake at room temperature for 2-3h, TBST After washing 3 times, add AP-labeled rabbit anti-mouse secondary antibody diluted 1:5000, shake at room temperature for 1 hour, wash 3 times with TBST, and use NBT / BCIP system to develop color. Observation of specific protein bands indicated that Penton protein had been expressed. (Such as Figure 6 sho...

Embodiment 3

[0159] Example 3: Composition, preparation and detection of Ankara subunit vaccine oil emulsion

[0160] Antigen: 500μg / ml purified target protein Penton

[0161] Adjuvant: ISA 71 VG water-in-oil (W / O)

[0162] Mass ratio of antigen to adjuvant: 3:7

[0163] Vaccine preparation: Dilute the purified target protein Penton to 500 μg / ml and add it to the container, then drop the adjuvant ISA 71 VG into the container according to the above ratio for emulsification.

[0164] Vaccine property test: after emulsification, the vaccine is dripped into clear water, the first drop will disperse, and the second drop will be in the form of oil beads, and the oil beads on the liquid surface will not break the emulsification.

[0165] Sterility test: Take a small amount of the vaccine and coat it on a TSA plate, and incubate in a 37°C incubator for 18 hours without colonies growing.

[0166] Stability test: Centrifuge at 3000rpm / min for 15 minutes, the emulsified vaccine is not separated af...

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Abstract

The invention discloses a preparation method and application of virus Ankara subunit vaccine. The preparation method is characterized in that recombinant plasmid pFastBac HTB-Penton transforming DH10Bac E.coli is constructed through a baculovirus expression system so as to obtain correct recombinant bacmid Bacmid-Penton, then Sf9 cells are transfected to obtain an immunogenic gene Penton, Fiber 2 in which recombinant virus Ac-Penton expresses virus Ankara; the increased expression and purifying of protein are carried out; the protein expression quantity is large, and the protein activity is high; the protecting rate of Ankara subunit vaccine using Penton protein as antigen and Ankara mixed subunit vaccine using Penton+Fiber 2 protein as the antigen to virus Ankara reaches 100%, and the antibodies produced by the two vaccines are in high level and can be maintained for a long time; virus Ankara can be treated effectively by the two vaccines, so that huge economic loss of virus Ankara infection to the breeding industry can be decreased.

Description

technical field [0001] The invention relates to the technical field of vaccine preparation, in particular to a preparation method and application of an Ankara virus subunit vaccine. Background technique [0002] In October 2015, Ankara virus broke out in Shandong, Anhui, Henan and other places in my country. The disease is caused by adenovirus and mainly causes nephritis, inclusion body hepatitis, pericardial effusion, and egg production drop syndrome. Adenoviruses are divided into subtypes I, II, and III. Subtype I is uncommon, subtype II (turkey hemorrhagic enteritis and related viruses), and subtype III is the recent buzz about nephritis, egg production decline syndrome, and tolerance. Body hepatitis and hydropericardium syndrome (also known as Ankara disease because it first broke out in Ankara, Pakistan). The disease occurs in broiler chickens aged 3 to 6 weeks, and the diseased chickens begin to die when they are more than 3 weeks old, reaching a peak at 4 to 5 weeks ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/66C12P21/02A61K39/235A61P31/20
CPCC12N15/86A61K39/12A61K2039/552A61K2039/55566C07K14/005C12N15/66C12N2710/10022C12N2710/10034C12N2710/14043C12N2800/105
Inventor 金梅林张丹邓雪霞马季康超杨影魏燕鸣张强孙小美
Owner HUAZHONG AGRI UNIV
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