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Goose astrovirus-like particle novel genetic engineering subunit vaccine

An astrovirus, amino acid technology, applied in genetic engineering, viruses, viral peptides, etc., can solve the problems of weak immune protection, strong strain virulence, incomplete inactivation, etc., to achieve strong immunogenicity, protein Good immunogenicity and the effect of reducing production costs

Active Publication Date: 2019-08-16
苏州世诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, antiviral and antibacterial treatment methods for goose astrovirus are ineffective, and there is no vaccine to prevent it; conventional attenuated live vaccines have the risk of returning to stronger virulence, and inactivated vaccines have weak autoimmune protection and inactivated inactivation. Completely, cause the risk of loose poison, therefore, prior art urgently needs a kind of novel genetic engineering vaccine of safe high-efficiency prevention goose Astrovirus, and the present invention therefore comes

Method used

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  • Goose astrovirus-like particle novel genetic engineering subunit vaccine
  • Goose astrovirus-like particle novel genetic engineering subunit vaccine
  • Goose astrovirus-like particle novel genetic engineering subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1 Construction and Identification of Transfer Vector pF-Cap

[0110] 1. Cap protein gene amplification and purification The codon-optimized GAstV Cap protein gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Co., Ltd. and cloned into the pUC17 plasmid to obtain the pUC-Cap plasmid vector. Use the pUC-Cap plasmid as a template, and Cap-F and Cap-R as upstream and downstream primers for PCR amplification (the gene sequences of Cap-F and Cap-R are shown in SEQ ID NO.3 and 4), and the amplification system is shown in the table 1.

[0111] Table 1 Cap protein gene amplification system

[0112]

[0113]

[0114] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0115] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the target band appea...

Embodiment 2

[0127] Embodiment 2 recombinant baculovirus genome Bac-Cap construction

[0128] 1. Transformation of DH10Bac bacteria Take 1 μl of the pF-Cap plasmid in Example 1 and add it to 100 μl of DH10Bac competent cells, mix well, bathe in ice for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, and then bathe in ice for 2 minutes, add 900 μl without Amp LB liquid medium, cultured at 37°C for 5 hours. After 100 μl of bacterial solution was diluted 81 times, 100 μl of diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and incubated at 37°C for 48 hours.

[0129] 2. Select a single clone Use an inoculation needle to pick up large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and culture at 37°C for 48 hours. Then pick a single colony and inoculate it into LB liquid medium containing gentamicin, kanamycin and tetracycline for culture, pres...

Embodiment 3

[0130] Example 3 Recombinant Baculovirus Transfection

[0131] Inoculate 0.8×10 per well in a six-well plate 6 Sf9 cells, the cell confluence is 50-70%. Prepare the following complexes for each well: dilute 4 μl of Cellfectin transfection reagent with 100 μl of transfection medium T1, and briefly vortex; dilute 3 μg of the recombinant Bacmid-Cap plasmid in Example 2 with 100 μl of transfection medium T1, Prepare the transfection mixture by mixing the diluted transfection reagent and plasmid separately and blowing gently. Add the above-mentioned transfection complex after the cells adhere to the wall, incubate at 27°C for 5 hours, remove the supernatant, add 2ml of SF-SFM fresh medium, culture at 27°C for 4-5 days, and harvest the supernatant. The recombinant baculovirus rBac-Cap was obtained, and the virus titer was detected by the MTT relative potency method for the harvested P1 generation recombinant baculovirus, and the rBac-Cap P1 virus titer was 3.4×10 7 pfu / mL. The a...

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Abstract

The invention discloses an immune composition which comprises goose astrovirus outer capsid structure protein coded by adopting nucleic acid molecules with a sequence shown as SEQ ID NO:1 or higher than 95% identical nucleic acid molecules of a nucleotide sequence shown as SEQ ID NO:1. The immune composition can be applied as a goose astrovirus-like particle novel genetic engineering subunit vaccine, is high in antigen expression level, can be spontaneously assembled into virus-like particles (VLPs) and is high in immunogenicity and apathogenic to gooses. The vaccine can be prepared on a largescale by using a bioreactor through serum-free suspended culture, and vaccine production cost is lowered greatly.

Description

technical field [0001] The invention relates to the technical field of animal immune drugs, in particular to a novel genetically engineered subunit vaccine of goose astrovirus virus-like particles. Background technique [0002] Astrovirus (Astroviridae, AstV) is a kind of non-enveloped, spherical, single-stranded positive-sense RNA virus with a diameter of 28-30nm. Astroviridae is divided into two genera, Mamastrovirus and Avastrovirus, which can cause a variety of diseases in poultry. Goose Astrovirus (Goose AstV, GAstV) belongs to avian Astrovirus type 3. The infection of this virus has a certain prevalence, and it mainly injures young goslings, mainly 5-20 days old. After geese are infected with GAstV, the incidence rate is as high as 80-90%, and the course of disease lasts for 7-10 days. The main symptom is goose goose, and the mortality rate is 20-70%, which causes serious economic losses to the goose breeding industry. [0003] At present, antiviral and antibacterial...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/14C07K14/08C12N15/40C12N15/866
CPCA61K39/12A61K39/39C07K14/005C12N15/86A61P31/14C12N2770/12022C12N2770/12034C12N2710/14043A61K2039/5258A61K2039/5256
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州世诺生物技术有限公司
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