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Recombinant beta-2 protein for inhibiting clostridium perfringens infection and preparation method and application thereof

A clostridium perfringens, protein technology, applied in antibacterial immunoglobulins, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as difficulty in increasing the renaturation rate

Inactive Publication Date: 2016-10-12
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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  • Recombinant beta-2 protein for inhibiting clostridium perfringens infection and preparation method and application thereof
  • Recombinant beta-2 protein for inhibiting clostridium perfringens infection and preparation method and application thereof
  • Recombinant beta-2 protein for inhibiting clostridium perfringens infection and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Soluble expression of β2-hisY

[0089] 1. Synthetic genes

[0090] The present application designed three kinds of recombinant β2 genes, namely β2-hisY gene shown in SEQ ID No.1, β2-hisW gene shown in SEQ ID No.3, and pmβ2-hisW gene shown in SEQ ID No.4.

[0091] Both the β2-hisY gene and the β2-hisW gene encode the protein β2-his shown in SEQ ID No.2. The pmβ2-hisW gene encodes the protein pmβ2-hisW shown in SEQ ID No.5. β2-his is a protein obtained by deleting amino acid residues 52-80 of pmβ2-hisW.

[0092] Synthesize the β2-Y gene shown in the 151-861 of SEQ ID No.1 (coding the protein shown in the 51-286 amino acid residues of SEQ ID No.2) by chemical synthesis, SEQ ID No .3 the β2-W gene shown in No. 151-861 (coding the protein shown in No. 51-286 amino acid residues of SEQ ID No.2), shown in No. 151-948 of SEQ ID No.4 pmβ2-W gene (encodes the protein pmβ2-W represented by amino acid residues 51-315 of SEQ ID No.5).

[0093] 2. Construction of reco...

Embodiment 2

[0113] Embodiment 2, animal immunoprotective test of β2-his

[0114] 1. Preparation of anti-Clostridium perfringens vaccine

[0115] The β2-his protein purified by molecular sieves in Example 1 was dissolved in sterile PBS to obtain a β2-his solution with a β2-his concentration of 1000 μg / mL for immunization. The β2-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 β2-his solution and incomplete Freund's adjuvant were mixed in an equal volume of 1:1, and emulsified to prepare an oil emulsion vaccine, which was named the second-immunity vaccine.

[0116] 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 strain C60-11. In the u...

Embodiment 3

[0135] Example 3, optimization of expression conditions induced by β2-his

[0136] 1. Optimization of induction temperature and time

[0137] Inoculate BL21(DE3) / pET30a-β2-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 to obtain culture medium) at 37°C, using a Thermo MaxQ6000 full-temperature shaker at 200rpm to shake and cultivate 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 i...

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Abstract

The invention discloses recombinant beta-2 protein for inhibiting clostridium perfringens infection and a preparation method and application thereof. The recombinant beta-2 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.286 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 recombinant beta-2 protein enables animals to have a higher serum antibody level and resist the attack of clostridium perfringens after the animals are immunized with the recombinant beta-2 protein. The recombinant beta-2 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 recombinant β2 protein for inhibiting Clostridium perfringens infection in the field of biotechnology, a preparation method and application thereof. 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. β2 toxin is a very impo...

Claims

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Application Information

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IPC IPC(8): C07K14/33C12N15/31C12N15/70C12N15/82C12N15/85C12N5/10A61K39/08A61P31/04G01N33/68G01N33/569C07K16/12
CPCA61K39/08C07K14/33C07K16/1282G01N33/569G01N33/68
Inventor 宋晓晖孙雨翟新验董浩
Owner CHINA ANIMAL DISEASE CONTROL CENT
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