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Sheep echinococcosis infection-resistant gene engineering subunit vaccine as well as preparation method and application thereof

A subunit vaccine and genetic engineering technology, applied in the field of genetically engineered subunit vaccines against sheep echinococcosis infection and its preparation, can solve the problems of high production costs, increased antigen production costs, and restrictions on popularization and application. Good immune protection, good solubility, and the effect of improving gene structure

Active Publication Date: 2018-05-25
SA BIOTECH (SUZHOU) PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When preparing vaccines, it still needs to be dissolved with high-concentration urea. Due to repeated denaturation and renaturation, it not only reduces its antigenicity but also greatly increases the production cost of antigens.
In order to improve its immunogenicity, Quil-A also needs to be used as an adjuvant. Therefore, each dose of vaccine needs up to 50 μg of antigen, resulting in high production costs, which greatly limits its application (Lightowlers, Parasitology, 2006, S27 -S42)

Method used

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  • Sheep echinococcosis infection-resistant gene engineering subunit vaccine as well as preparation method and application thereof
  • Sheep echinococcosis infection-resistant gene engineering subunit vaccine as well as preparation method and application thereof
  • Sheep echinococcosis infection-resistant gene engineering subunit vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction of recombinant expression vector of pET28b-2EG95 subunit vaccine against echinococcosis infection

[0037] 1.1 Configuration of solution and medium

[0038] Solution I: 25mmol / L Tris-HCl (pH8.0), 10mmol / L EDTA.

[0039] Solution II: 0.2mol / L NaOH, 1% SDS (prepared and used immediately).

[0040] Solution III: 100mL system, 80mL 5mol / L potassium acetate, 12mL glacial acetic acid, 8mL ddH2O.

[0041] TAE (50×): 242g Tris base, 57.1mL glacial acetic acid, 100mL0.5mol / LEDTA (pH8.0), dilute to 1000mL with sterile water.

[0042] LB medium: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, adjust the pH value to 7.0 (solid medium contains 1.5% agar).

[0043] Protein loading buffer (2×): 100mmol / L Tris-HCl (pH6.8), 200mmol / L dithiothreitol (DTT), 4% SDS, 0.2% bromophenol blue, 10% glycerol.

[0044] 29 g of 30% acrylamide solution: acrylamide, 1 g of N, N'-methylene acrylamide, dissolved in 100 mL of water, filtered.

[0045] Coomassie bri...

Embodiment 2

[0073] Example 2, pET28b-2EG95 recombinant plasmid induces expression in Escherichia coli BL21 (DE3)

[0074] Transform the pET28b-2EG95 recombinant plasmid into BL21(DE3) Escherichia coli, and spread the LB solid plate (the method is the same as "1.5"). Through monoclonal screening, high-quality and high-yield strains were obtained. Then inoculate the high-yield strain into 500mL LB medium (containing ampicillin and chloramphenicol), and culture it with shaking at 37°C until the OD value is about 1.5-2.0, then inoculate the seed liquid into the fermenter for For fermentation, when the OD value of the bacteria reaches about 25-30, the temperature is lowered to 20°C, and when the temperature is stable, IPTG is added to a final concentration of 0.4mM, and induced for 12-16 hours.

Embodiment 3

[0075] Embodiment 3, the purification of hydatid hydatid 2EG95 protein

[0076] Buffer Z: 25mM Tris-HCl (pH7.2), 25mM Potassinm Phosphate (pH6.8), 500mM NaCl, 10% Glycerol, 1mM DTT

[0077] Equilibrium buffer: 10mM Tris-HCl (pH7.2), 250mM NaCl, 10% Glycerol

[0078] 1M imidazole, pH 7.0

[0079] 3.1 Fermentation broth centrifugation and whole cell disruption

[0080] After the fermentation, the fermentation broth was centrifuged (10,000 rpm for 8 min), the supernatant was discarded, and the precipitate was resuspended with BufferZ. The resuspended bacteria were crushed with a high-pressure homogenizer (crushing pressure: 1000 bar), centrifuged after crushing (23000 rpm for 1 h), and the supernatant was collected.

[0081] 3.2 Purification of target protein

[0082] Add 3-5% imidazole to the crushed supernatant, first use 100% Equilibrium buffer to equilibrate the Ni-NTA chromatography column, then hang the sample, and directly use 25% imidazole to elute the target protein ...

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Abstract

The invention discloses a preparation method of sheep echinococcosis infection-resistant gene engineering subunit vaccine. The preparation method comprises the following steps: S1, searching and downloading EG95 gene sequence as shown in SEQ ID NO.2 as well as EG95 amino acid sequence as shown in SEQ ID NO.3 of echinococcus granulosus from NCBI and performing amino acid sequence modification to obtain the modified EG95 amino acid sequence; S2, performing tandem expression on the gene sequences of a plurality of modified EG95 amino sequences through flexible linker to form recombinant multi-EG95 gene sequence; S3, cloning the recombinant multi-EG95 gene sequence to a pET28b plasmid vector, converting to Escherichia coli BL21 (DE3), and performing induction expression by a tag added fusion expression mode to obtain recombinant protein; and S4, performing protein purification on the recombinant protein to obtain the sheep echinococcosis infection-resistant gene engineering subunit vaccine. According to the preparation method, the production cost of echinotype antigen can be greatly reduced, the production process is greatly simplified and various advantages of safety, high efficiency,low cost and the like are achieved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a genetic engineering subunit vaccine against sheep echinococcosis infection and its preparation method and application. Background technique [0002] Hydatid disease, also known as echinococcosis, is a zoonotic disease caused by the larvae of the genus Echinococcus (Genus echinococcus). Echinococcus species include Echinococcus granulosus (Echinococcus granulosus, Eg), Echinococcus multilocularis (E.multilocularis), Echinococcus fowleri (E.vogeli) and Echinococcus oligarthrus (E.oligarthrus), etc. , whose morphology, host and distribution area are slightly different, and Echinococcus granulosus is the most common. Echinococcus granulosus belongs to the flatworm phylum, cestodes, multi-section subclass, round leaf order, ribbon family, echinococcus genus, and its larvae are called hydatidcyst, which are round or irregular cysts , composed of cyst wall, germinal cyst...

Claims

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Application Information

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IPC IPC(8): A61K39/00C12N15/12C12N15/70C07K14/435A61P33/10
CPCA61K39/00A61K2039/552C07K14/43554C12N15/70
Inventor 袁于人殷波陆洲
Owner SA BIOTECH (SUZHOU) PTE LTD
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