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Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli

一种猪丹毒杆菌、保护性抗原的技术，应用在抗体医疗成分、化学仪器和方法、细菌肽等方向，能够解决不容易回收和纯化、很难畜牧业者接受等问题

Active Publication Date: 2007-09-05
MEIJI ANIMAL HEALTH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Efficient recovery and purification of the desired soluble SpaA protein from this multi-contaminant mixture is not easy
Generally, animal vaccines are different from human vaccines, unless they are cheap, high-quality and high-purity, they are difficult to be accepted by livestock farmers

Method used

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  • Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli
  • Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli
  • Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] (1) Cloning of the gene encoding SpaA protein or ΔSpaA protein

[0097] At 37°C, type 1 strains Fujisawa strain and Koganei strain and type 2 strain Tama 96 strain and SE-9 strain 16 -48 hours. Centrifuge the cultured bacteria solution (about 1.5-3.0 mL), and extract the whole genome DNA from the obtained precipitate (about 0.03 g or more) with a DNA extraction kit (Isoplant, Nippon Genes Co., Ltd.).

[0098] With this whole genome DNA as template, use the synthetic primer (SEQ ID NO:3 and 4 sequence pair, SEQ ID NO:3 and 5 sequence pair) prepared based on nucleotide sequence SEQ ID NO:1 and LAPCR kit (Baobao wine) for PCR. The reaction solution was kept at 94°C for 3 minutes, and then circulated at 94°C-60 seconds, 56°C-30 seconds and 72°C-60 seconds, repeating 30 cycles. Primer SEQ ID NO: 3 was designed to amplify the downstream region from the 79th nucleotide of the SpaA gene, and an NcoI site was added to its 5' end. Primers SEQ ID NO: 4 and 5 were designed to a...

Embodiment 2

[0109] (1) Expression of protein in the form of inclusion body caused by amino acid substitution of ΔSpaA protein

[0110] The plasmid construction method is as follows: use appropriate restriction enzymes to cut the plasmid from the clone that forms the inclusion body in Example 1-(4), and insert the obtained DNA fragment containing the nucleotide substitutions described in Table 2 into the DNA fragment encoding the expression of soluble ΔSpaA from The corresponding region in the gene of the ΔSpaA protein in the plasmid extracted from the clone of the protein (SE-9 strain).

[0111] specifically,

[0112] (a) After the plasmid from clone No.1 was double-digested with restriction enzymes EcoI and ClaI, agarose electrophoresis was performed to separate the EcoI-ClaI fragment (Fig. 4A-1), which contains the 587th-position in the gene encoding ΔSpaA protein The sequence of the 1152nd nucleotide. The resulting fragment was inserted into a plasmid from a clone expressing soluble ...

Embodiment 3

[0127] (1) Purification of SpaA protein and ΔSpaA protein forming inclusion bodies

[0128] Each of the Escherichia coli cells expressing the ΔSpaA protein in the form of inclusion bodies obtained in Example 2-(1) and the Escherichia coli cells expressing the SpaA protein in the form of inclusion bodies obtained in Example 2-(2) were cultured. Take 100ml of the culture solution, centrifuge at 10,000rpm for 5 minutes, and add 1 / 5-1 / 10 volume washing buffer (20mM Tris-Hcl pH7.5, 10mM EDTA, 1% Triton X-100), suspend the cells until uniform. Add 1 / 100 volume of lysozyme solution (10 mg / ml) to the suspension, and react at 30° C. for 15 minutes. Under ice-cold conditions, sonicate the mixture with a sonication cell homogenizer (manufacturer: Barnson Sonic Power; model: 350; output: 4; duty cycle: 30%; time: 5-15 minutes) and centrifuge at 15,000rpm 15 minutes. After recovering the supernatant, add the same volume of wash buffer (or sterile distilled water) to the pellet as the so...

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Abstract

It is intended to provide a mutant of a surface protective antigen SpaA protein of Erysipelothrix rhusiopathiae or a shortened SpaA protein (DeltaSpaA) obtained by removing a part therefrom and a process for producing the same. By introducing an amino acid substituent into a definite position of the amino acid sequence of the SpaA protein or the DeltaSpaA protein, a mutant of the SpaA protein or the DeltaSpaA protein having immunogenicity and being expressed as an inclusion body in Escherichia coli can be obtained. Because of being expressed as an insoluble inclusion body in Escherichia coli, the above-described mutant of the SpaA protein or the DeltaSpaA protein can be easily collected and purified.

Description

technical field [0001] The invention relates to a method for preparing Erysipelas suis surface protective antigen (hereinafter also referred to as "SpaA") variant by using E. coli (Escherichia coli) as a host. More specifically, the present invention relates to a method for preparing a SpaA variant or a shortened SpaA (hereinafter also referred to as "ΔSpaA") variant obtained by removing a part of SpaA by introducing an amino acid substitution, wherein the variant is expressed in Escherichia coli cells When expressed in vivo, it can be expressed in the form of insoluble inclusion bodies; and variants involving recombinant SpaA or ΔSpaA protein obtained by this method. Background technique [0002] Erysipelas is a pig disease caused by Erysipelothrix rhusiopathiae. Infected pigs show symptoms such as acute sepsis, subacute urticaria, or chronic endocarditis and arthritis. According to reports, about 3,000 pigs fall ill each year, causing huge losses to livestock farmers. Er...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/195C12P21/02A61K38/16A61P31/00A61K38/00A61K39/00
CPCC07K14/195A61K38/00A61K39/00A61P19/02A61P31/00A61P31/04A61P37/04A61P43/00A61P9/00
Inventor 牛岛稔大坂口正士德永英治
Owner MEIJI ANIMAL HEALTH CO LTD
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