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Preparation method of fusion protein inhibiting Clostridium perfringens infection

A protein and coding gene technology, which is applied in the field of preparation of fusion proteins for inhibiting Clostridium perfringens infection, and can solve the problems of difficulty in increasing the renaturation rate and the like

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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  • Preparation method of fusion protein inhibiting Clostridium perfringens infection
  • Preparation method of fusion protein inhibiting Clostridium perfringens infection
  • Preparation method of fusion protein inhibiting Clostridium perfringens infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Soluble expression of α-β2-ε-his

[0051] 1. Synthetic genes

[0052] The application 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. The pmα-β2-ε-hisW gene indicated.

[0053] 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, 464-492 and 743-750 of pmα-β2-ε-hisW.

[0054] The α-β2-ε-Y gene shown in the 151-2814th position of SEQ ID No.1 is synthesized by chemical synthesis (the protein shown in the 51-937th amino acid residue of encoding SEQ ID No.2) , the α-β2-ε-W gene shown in the 151-2814th position of SEQ ID No.3 (the protein shown in the 51-937th amino acid residue of encoding SEQ ID No.2), the The pmα-β2-ε-W gene represented...

Embodiment 2

[0075] Embodiment 2, animal immune protective test of α-β2-ε-his

[0076] 1. Preparation of anti-Clostridium perfringens vaccine

[0077] The α-β2-ε-his protein purified by molecular sieves 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 emulsified to prepare an oil emulsion vaccine, which was named the first vaccine. The α-β2-ε-his solution and incomplete Freund's adjuvant were mixed in equal volumes of 1:1, and emulsified to prepare an oil emulsion vaccine, which was named as the secondary vaccine.

[0078] Take out the A-type Clostridium perfringens virulent strain C57-10, the B-type Clostridium perfringens virulent strain C58-5, and the C-type Clostridium perfringens virulent strain purchased from the China Veterinary Drug Administration. Strain C59-4, Clostridium perfringens type D virulent st...

Embodiment 3

[0101] Example 3, Optimization of α-β2-ε-his Induced Expression Conditions

[0102] 1. Optimization of induction temperature and time

[0103] Inoculate BL21(DE3) / pET30a-α-β2-ε-Y in LB liquid medium containing 50 μg / ml kanamycin (add kanamycin to LB liquid medium until the concentration of kanamycin is 50 μg / ml obtained medium), 37 ° C, using Thermo MaxQ6000 type full-temperature shaker 200 rpm shaking culture to OD 600 When the value (the LB liquid medium containing 50 μg / ml kanamycin was used as the blank control) reached 0.6, isopropylthio-β-D-galactoside (IPTG) was added to induce the following six kinds of expression respectively. The first induced expression was induced with 0.75 mM IPTG for 1 hour at 37°C. The second induced expression was induced with 0.75 mM IPTG for 2 hours at 37°C. The third induced expression was induced with 0.75 mM IPTG for 4 hours at 37°C. The fourth induced expression was induced with 0.75 mM IPTG for 5 hours at 37°C. The fifth induced exp...

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Abstract

The invention discloses a preparation method of fusion protein for inhibiting clostridium perfringens infection. The method comprises the step that encoding genes of protein are expressed in organisms to obtain the fusion protein, wherein the organisms are microorganisms or plants or non-human animals; the 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 prepared through the method 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 preparation method of a fusion protein inhibiting Clostridium perfringens infection in the field of biotechnology. Background technique [0002] Clostridium perfringens (Clostridium Perfringens), also known as Clostridium welchii, is an important zoonotic disease. The pathogen is traumatic gas gangrene and human food poisoning, as well as sheep blight, lamb dysentery, and necrosis of cattle and sheep. It is one of the main pathogens of acute enteritis and cattle and sheep enterotoxemia, which has caused huge economic losses to animal husbandry. The main pathogenic factor of Clostridium perfringens is the exotoxin secreted by it. There are as many as 13 types, among which α, β and ε are the most important exotoxins. According to the different types of exotoxins produced, the perfringens can be Clostridium mycobacterium is divided into five serotypes A, B, C, D, and E. At present, immunization against Clostridium perfringens ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/116A61K39/08A61P31/04
CPCA61K39/08C07K14/33C07K2319/00C07K2319/21C12N15/70C12N2800/101
Inventor 宋晓晖孙雨翟新验董浩
Owner CHINA ANIMAL DISEASE CONTROL CENT
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