I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof

A subunit vaccine and poultry adenovirus technology, applied in the field of vaccines, can solve the problems of lack of subunit vaccines, unreasonable selection of antigens, poor immune effect, etc.

Inactive Publication Date: 2017-01-25
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the technical defects of the prior art, and provides a genetically engineered subunit vaccine of group I adenovirus type 4 and its preparation method, so as to solve the difficulty in preparing the inactivated vaccine of group I adenovirus type 4 in the prior art technical issues
[0008] Another technical p

Method used

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  • I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof
  • I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof
  • I-group 4-type aviadenovirus genetic engineering subunit vaccine and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 (fibrin C-terminal gene cloning and sequence analysis)

[0035] Design and synthesize a pair of primers:

[0036] Primer 1: 5'-GCGAATTCATGGCTATGCTACAGAT-3';

[0037] Primer 2: 5'-GGAAGCTTTTACGGGAGGGAGCC-3'.

[0038] FAV-4 viruses were purchased from China Veterinary Drug Control Institute.

[0039] Using the FAV-4 virus as a template, denaturation at 94°C for 45s, annealing at 56°C for 45s, and extension at 72°C for 1min, a total of 30 cycles of PCR amplification were performed; the PCR product was directly cloned into the pMD-18T vector (TaKaRa Company) to construct pMD-18T-HHS; Sequencing was carried out by the dideoxy chain termination method, and the results showed that the gene encoding the fibrin C-terminus cloned from the FAV-4 genome had the nucleotide sequence shown in SEQ ID No: 2, which encoded The amino acid sequence is shown in SEQ ID No:1.

Embodiment 2

[0040] Embodiment 2 (construction of expression vector and engineering bacterium)

[0041] Digest pMD-18T-HHS and pET-32a with EcoR I and Hind III, perform 1% agarose gel electrophoresis on the digested products, recover the target fragment and pET-32a respectively, and then connect overnight at 16°C; Transformed into E.coliDH5α competent cells, picked a single colony and inoculated it into 2ml LB medium containing 100μg / mL ampicillin, cultured with shaking at 37°C for 12h, extracted the plasmid and identified it by double enzyme digestion, and obtained the expression plasmid pET-32a- HHS. Transform the recombinant plasmid into E.coliBL21(DE3), pick a single colony, inoculate into 200mL LB medium containing 100μg / mL ampicillin, culture with shaking at 37°C for 12h, then transfer to 4L of the same medium, shake at 37°C After culturing for 3 hours, IPTG was added to a final concentration of 0.5 mM, and culture was continued for 4 hours, followed by SDS-PAGE electrophoresis anal...

Embodiment 3

[0042] Example 3 (purification of the C-terminus of the fibrous protein of the recombinant group I group 4 avian adenovirus)

[0043] Pick a single colony of engineering bacteria in 200ml LB liquid medium (containing ampicillin at a concentration of 0.1g / L), and cultivate overnight at 37°C with constant temperature shaking; pour the overnight culture into a total of 4L of LB liquid at a volume ratio of 1:20 Culture medium (ampicillin concentration: 0.1 g / L) was cultured at 37°C with constant temperature and shaking for 3 hours; then IPTG was added to make the final concentration 5 mM, and cultured at 37°C with constant temperature and shaking for 4 hours.

[0044] Centrifuge the culture with a large-scale low-temperature refrigerated centrifuge, 8000rpm, 20min; discard the supernatant, add 100ml of binding buffer (20mmol / LTris.HCl, pH 8.0, 0.5mol / L NaCl, 5mmol / L imidazole) to suspend the cells, and then Perform 750W ultrasonic crushing (working time 10s, interval time 20s, cru...

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Abstract

The invention provides an I-group 4-type aviadenovirus genetic engineering subunit vaccine and a preparation method thereof. According to the technical scheme, the preparation method comprises the following steps: cloning an encoding gene of fibrous protein C-terminal from an I-group 4-type aviadenovirus genome according to a PCR technology and performing sequence analysis; cloning the gene to an expression vector pET-32a, transforming escherichia coli, constructing engineering bacteria, and inducing the engineering bacteria by isopropyl-beta-D-thiogalactopyranoside to express the fibrous protein C-terminal; performing lysis on an engineering bacterial cell, performing centrifugal separation on an inclusion body of the engineering bacterial cell, dissolving urea and diluting for renaturation; preparing the vaccine according to the conventional preparation method of a mineral oil adjuvant inactivated vaccine. According to the I-group 4-type aviadenovirus genetic engineering subunit vaccine prepared by the method, the immune effect of the vaccine is evaluated by a serological method and an immunity challenge method, and the result indicates that the aviadenovirus inactivated vaccine prepared by the method can provide effective immunoprotection and has a good commercialized development prospect.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and at the same time relates to the preparation of recombinant bacteria and the expression of recombinant proteins, in particular to a genetically engineered subunit vaccine of group I type 4 avian adenovirus and a preparation method thereof. Background technique [0002] Group I avian adenoviruses make up the genus Avian Adenovirus in the family Adenoviridae. Group II (Turkey Hemorrhagic Enteritis and Related Viruses) and Group III (Egg Drop Syndrome) adenoviruses are clearly associated with disease, in contrast to most Group I avian adenoviruses that have not been shown to be pathogenic in poultry. totally sure. But FAdV-1 and FAdV-4 are obviously exceptions, among which FAdV-1 can cause quail bronchitis, and FAdV-4 is the main cause of hydropericardium hepatitis syndrome. Other adenovirus strains allow rapid infection of opportunistic pathogens when chicken health is compromised, such as co...

Claims

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Application Information

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IPC IPC(8): A61K39/235A61P31/20C12N15/70C12N15/34
CPCA61K39/12A61K2039/5252A61K2039/552C07K14/005C12N15/70C12N2710/10222C12N2710/10234
Inventor 唐宏荣李亚杰杨保收付旭彬李守军
Owner TIANJIN RINGPU BIO TECH
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