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Immune protective antigen of haemophilus parasuis

A technology of Haemophilus suis and antigenic protein, which is applied in the field of subunit vaccine preparation of animal infectious diseases, can solve the problems affecting the control effect of commercial vaccines, Haemophilus parasuis is not, is not, etc.

Active Publication Date: 2013-01-09
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commercial inactivated vaccines and self-made vaccines can control the infection of Haemophilus parasuis to a certain extent, but the diversity of serotypes and a large proportion of strains that cannot be typed affect the control effect of commercial vaccines, so it is often used on a large scale. Failed to immunize
The protection of home-made vaccines may be more targeted, but there are many examples of failure, mainly because the isolated Haemophilus parasuis is not the real pathogenic strain or the main pathogenic serotype

Method used

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  • Immune protective antigen of haemophilus parasuis
  • Immune protective antigen of haemophilus parasuis
  • Immune protective antigen of haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Cloning and expression of Haemophilus parasuis recombinant antigenic protein Hbp B

[0022] a material

[0023] 1 Plasmids and strains

[0024] The pET-28a (+) plasmid used in the present invention is purchased from Novagen, Germany, and the pET-28a (+) plasmid map is as follows Figure 7 shown. Competent cells Escherichia coli DH5α and BL 21 (DE 3) are products of Beijing Quanshijin Biotechnology Co., Ltd.

[0025] The Haemophilus parasuis bacterial strain used in the present invention is the HPS SH0165 bacterial strain and screens for the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University (referring to: Cai Xuwang, the research on the separation and identification of Haemophilus parasuis and the diagnostic method and inactivated vaccine, 2006 6 Month, Ph.D. Dissertation of Huazhong Agricultural University, National Library of China, National Digital Library of China http: / / res4.nlc.gov.cn / home / search.trs?method=showDeta...

Embodiment 2

[0113] Example 2: Characteristic Analysis of Recombinant Antigen Protein

[0114] 1 Analysis of the characteristics of the recombinant antigenic protein by Western-blot method

[0115] The recombinant antigen protein Hbp B refolded and purified in Example 2 was subjected to SDS-PAGE gel electrophoresis according to conventional methods. Subsequent steps are as follows:

[0116] 1) Transfer membrane: Cut out 6 pieces of Whatman 3M filter paper and 1 piece of nitrocellulose membrane (NC membrane). The size of the filter paper and NC membrane should be completely equal to or slightly smaller than the gel, and mark the corner of the NC membrane with a pencil , determine the relative direction of the membrane and the gel after transfer; soak the NC membrane in purified water for 5 min; add a small amount of transfer buffer to another shallow tray, and soak 6 layers of Whatman 3M filter paper in it. Then install the transfer electrophoresis tank according to the following steps: l...

Embodiment 3

[0138] Embodiment 3: Recombinant antigen protein mouse immune protection test

[0139] 1 Mass expression and purification of recombinant proteins

[0140] A single colony containing the recombinant expression strain was inoculated into 10 mL of LB liquid medium containing 0.25 μg / mL kanamycin, and cultured on a shaker at 37°C. Inoculate the cultured bacteria solution into 1L of fresh LB liquid medium containing 2.5μg / mL kanamycin, shake and culture at 37°C for about 3h, until the OD 600 When it reaches 0.6-0.8, add IPTG to a final concentration of 0.8mmol / L, continue to induce for 3h, and then collect the bacteria. See Example 2 for the specific purification process of the recombinant protein.

[0141] 2 Determination of recombinant protein concentration (conventional Bradford method)

[0142] See embodiment 2 for specific operations.

[0143] 3 Preparation of recombinant antigenic protein vaccine and immunization method

[0144] 1) Preparation of recombinant antigen protei...

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Abstract

The invention belongs to the technical field of animal-borne disease subunit vaccine preparation and relates to preparation and application of the immune protective antigen of the haemophilus parasuis. Outer membrane protein Hbp B genes of the haemophilus parasuis are cloned, a nucleotide sequence is indicated as SEQID NO:1, and the sequence of gene code is indicated as SEQ ID NO:2. Recombination Escherichia coli BL21 / Pet-28a-Hbp B (preservation number is CCTCC NO:M2011228) is built and comprises genes in a sequence table SEQ ID NO:1. Antigen protein of the haemophilus parasuis is obtained and expressed through gene transformation Escherichia coli in the SEQ ID NO:1. The invention further discloses a preparation method and application of the recombination Escherichia coli. The haemophilus parasuis subunit vaccine has good safety, and an immune protection effect reaches 83%.

Description

technical field [0001] The invention belongs to the technical field of preparation of animal infectious disease subunit vaccines, and in particular relates to the preparation and application of a Haemophilus parasuis protective antigen. Aiming at the prevention and control of Haemophilus parasuis disease, the present invention designs the cloning expression, functional verification and vaccine application research of the gene Hbp B encoding a protective antigenic protein. The antigenic protein expressed by the gene can improve the ability of pigs to resist Haemophilus parasuis disease. Background technique [0002] Haemophilus parasuis (HPS) can cause Glasser's disease in pigs, which is characterized by fibrinous polyserositis, arthritis and meningitis. Currently, according to the agar diffusion serotyping method, Haemophilus parasuis can be divided into 15 standard serotypes (Kieletein et al., 1992), and more than 20% of clinical isolates cannot be typed. Among them, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/21C12N15/63C07K14/285A61K39/102A61P31/04C12R1/19
Inventor 金梅林周明光赵建平张强张安定康超徐高原
Owner HUAZHONG AGRI UNIV
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