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Fusion protein inhibiting Clostridium perfringens infection and related biomaterials and applications

A biological and protein technology, applied in the direction of biological material analysis, application, and analytical materials, can solve problems such as difficulty in increasing renaturation rate

Inactive Publication Date: 2020-01-14
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein denaturation and renaturation is an extremely complicated process. The renaturation conditions of different proteins are different, and the renaturation rate is often difficult to improve.
This is the main constraint limiting its application

Method used

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  • Fusion protein inhibiting Clostridium perfringens infection and related biomaterials and applications
  • Fusion protein inhibiting Clostridium perfringens infection and related biomaterials and applications
  • Fusion protein inhibiting Clostridium perfringens infection and related biomaterials and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1. Soluble expression of α-β2-ε-his

[0080] 1. Synthetic genes

[0081] This application has designed three fusion genes, which are the α-β2-ε-hisY gene shown in SEQ ID No. 1, the α-β2-ε-hisW gene shown in SEQ ID No. 3, and the α-β2-ε-hisW gene shown in SEQ ID No. 4, respectively. The pmα-β2-ε-hisW gene shown.

[0082] Both the α-β2-ε-hisY gene and the α-β2-ε-hisW gene encode the protein α-β2-ε-his shown in SEQ ID No.2. The pmα-β2-ε-hisW gene encodes the protein pmα-β2-ε-hisW shown in SEQ ID No.5. α-β2-ε-his is a protein obtained by deleting amino acid residues 52-146, amino acid residues 464-492, and amino acid residues 743-750 of pmα-β2-ε-hisW.

[0083] The α-β2-ε-Y gene shown at positions 151-2814 of SEQ ID No. 1 (encoding the protein shown at amino acid residues 51-937 of SEQ ID No. 2) was synthesized by chemical synthesis , The α-β2-ε-W gene shown in positions 151-2814 of SEQ ID No. 3 (encoding the protein shown in amino acid residues 51-937 of SEQ ID No. 2), o...

Embodiment 2

[0104] Example 2. Animal immune protection test of α-β2-ε-his

[0105] 1. Preparation of vaccine against Clostridium perfringens

[0106] The α-β2-ε-his protein purified by the molecular sieve in Example 1 was dissolved in sterile PBS to obtain an α-β2-ε-his solution with an α-β2-ε-his concentration of 1000 μg / mL for immunization. The α-β2-ε-his solution and Freund's adjuvant were mixed in an equal volume of 1:1, and then emulsified to prepare an oil emulsion vaccine, which was named the first vaccine. The α-β2-ε-his solution and the incomplete Freund's adjuvant were mixed in an equal volume of 1:1, and the oil emulsion vaccine was prepared by emulsification, which was named the second vaccine.

[0107] Take out the virulent Clostridium perfringens type A strain C57-10, the virulent Clostridium perfringens type B strain C58-5, and the virulent Clostridium perfringens type C purchased from the China Veterinary Drug Inspection Institute Strain C59-4, C60-11 virulent strain of Clostri...

Embodiment 3

[0130] Example 3. Optimization of α-β2-ε-his induced expression conditions

[0131] 1. Optimization of induction temperature and time

[0132] BL21(DE3) / pET30a-α-β2-ε-Y was inoculated into LB liquid medium containing 50μg / ml kanamycin (Kanamycin was added to the LB liquid medium to the concentration of kanamycin 50μg / ml culture medium), 37℃, using Thermo MaxQ6000 full temperature shaker 200rpm shaking culture to 0D 600 When the value (using LB liquid medium containing 50 μg / ml kanamycin as a blank control) reached 0.6, isopropylthio-β-D-galactoside (IPTG) was added to induce the expression of the following 6 kinds respectively. The first induction of expression was induced with 0.75 mM IPTG at 37°C for 1 hour. The second induction of expression was induced with 0.75 mM IPTG at 37°C for 2 hours. The third induced expression was induced with 0.75 mM IPTG at 37°C for 4 hours. The fourth induced expression was induced with 0.75mM IPTG at 37°C for 5 hours. The fifth induction of exp...

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Abstract

The invention discloses fusion protein for inhibiting clostridium perfringens infection and a related biological material and application thereof. The fusion protein is shown in (a) or (b) or (c), wherein the protein in (a) is composed of amino acid sequences shown as SEQ ID No.2; the protein in (b) is composed of amino acid sequences shown at the sites No.51-No.937 of SEQ ID No.2; the fusion protein in (c) is obtained by fusing protein tags at carboxyl terminals or / and amino terminals of the protein shown in (a) or (b). The fusion protein enables animals to have a higher serum antibody level and resist attack of clostridium perfringens after the animals are immunized with the fusion protein. The fusion protein is good in solubility and easy to purify and can serve as a diagnostic antigen to be prepared into a monoclonal antibody or be used for further research on protein functions and conformation relations.

Description

Technical field [0001] The invention relates to a fusion protein for inhibiting infection of Clostridium perfringens and related biological materials and applications in the field of biotechnology. Background technique [0002] Clostridium Perfringens (Clostridium Perfringens), also known as Clostridium wilfordii, is an important zoonotic disease. The pathogen is traumatic gas gangrene and human food poisoning, as well as fast epidemic of sheep, lamb dysentery, and necrosis of cattle and sheep. Enteritis, one of the main pathogens of enterotoxemia in cattle and sheep, has caused huge economic losses to the livestock industry. The main pathogenic factor of Clostridium perfringens is the exotoxin secreted by it. There are as many as 13 kinds of exotoxins. Among them, α, β and ε are the most important exotoxins. Clostridium hymenae is divided into five serotypes: A, B, C, D, and E. At present, the immunity against Clostridium perfringens is mainly achieved through traditional inac...

Claims

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Application Information

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IPC IPC(8): C07K14/195C07K19/00C12N15/31G01N33/68G01N33/569
CPCC07K14/195C07K2319/20C07K2319/40G01N33/56911G01N33/6854G01N2469/10
Inventor 宋晓晖孙雨翟新验曲萍
Owner CHINA ANIMAL DISEASE CONTROL CENT
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