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Preparation and application of GIT fusion protein used for preventing dairy cow mastitis

A technology of cow mastitis and fusion protein, which is applied in the direction of medical preparations containing active ingredients, hybrid peptides, and the use of carriers to introduce foreign genetic materials, etc., can solve the problem that the immune protection effect is not as good as that of fusion proteins, and achieve guaranteed Immunoprotective effect, simplified preparation process, shortened peptide chain length

Inactive Publication Date: 2012-10-17
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since cow mastitis is a multi-pathogenic disease, neither a single Staphylococcus aureus subunit vaccine nor a certain Streptococcus subunit vaccine can meet the clinical needs
For this reason, we tried to mix several subunit vaccines together for immunization to achieve the effect of one-shot immunization to prevent several bacterial infections. The research found that it may be the reason why the components of the subunit vaccine interfere with each other. The immunoprotective effect produced by mixing the subunit vaccines together is far less than the immune protective effect of the fusion protein of several antigens

Method used

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  • Preparation and application of GIT fusion protein used for preventing dairy cow mastitis
  • Preparation and application of GIT fusion protein used for preventing dairy cow mastitis
  • Preparation and application of GIT fusion protein used for preventing dairy cow mastitis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This example illustrates the construction of intermediate plasmids containing gapC1 and gapC2, the specific steps are as follows:

[0046] 1. According to the published gapC gene sequence, the gene is divided into two fragments, the sequences are shown in SEQ ID No.3 and SEQ ID No.4, and the amino acid sequences of gapC1 and gapC2 are shown in SEQ ID No.5 and SEQ ID As shown in No.6, use Oligo6.0 and Gene Runner software to design two pairs of PCR primers named F1, R1 (for amplifying the gapC1 gene fragment), and F2, R2 (for amplifying the gapC2 gene fragment). The codes, primer positions, sequences and amplified fragment lengths of the primers are listed in Table 1. The upstream primer used to amplify the gapC1 gene fragment introduces a BamH I restriction enzyme site, the downstream primer introduces a Hind III restriction enzyme site, and the upstream primer used to amplify the gapC2 gene fragment introduces a BamH I restriction enzyme The cleavage site, the Sal I r...

Embodiment 2

[0055] This example illustrates the construction of recombinant protein prokaryotic expression vectors gapC-pET32a, gapC1-pET32a, gapC2-pET32a, induction and purification of recombinant protein, and the specific steps are as follows:

[0056] 1. Use BamH I and Sal I to double-digest a part of pET32a for the connection of gapC and gapC2 gene fragments; use restriction endonucleases BamH I and Hind III to double-digest part of the pET-32a plasmid, and use Ligated to the gapC1 gene fragment, pET32a after enzyme digestion was recovered and purified;

[0057] 2. The recombinant cloning plasmid gapC1-pMD18 was double digested with restriction endonucleases BamH I and Hind III, and the recombinant cloning plasmids gapC2-pMD18 and gapC-pQE30 were digested with restriction endonucleases BamH Ⅰ and Sal Ⅰ respectively. Perform double enzyme digestion, and recover and purify the gapC1, gapC2, and gapC gene fragments after enzyme digestion;

[0058] 3. Ligate the gapC1, gapC2, and gapC ge...

Embodiment 3

[0061] This embodiment is used to compare the immunity containing gapC1, gapC2, gapC protein, and is divided into the following parts:

[0062] 1. Use the Western Blotting method to detect the immunogenicity of the recombinant protein (wet transfer method)

[0063] The purified GapC protein, GapC1 protein and GapC2 protein and the pre-stained protein Maker were subjected to Western Blotting respectively according to anti-serum of Streptococcus lactis (1:100), anti-serum of Streptococcus agalactiae (1:200), anti-serum of Streptococcus uberis Dilute the primary antibody with serum (1:100), and dilute the secondary antibody with HRP-labeled goat anti-mouse IgG (1:400in TBST). The result is as Figure 5 As shown, GapC protein, GapC1 protein and GapC2 protein can all react specifically with the antiserum of Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis, and specific bands appear at the corresponding positions of the NC membrane.

[0064] 2. Mice i...

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Abstract

The invention provides a GIT fusion protein used for preventing dairy cow mastitis. The GIT fusion protein has an amino acid sequence as represented by SEQ ID No. 1 or an amino acid sequence which is formed after substitution, deletion or addition of one or a plurality of amino acids of the sequence represented by SEQ ID No. 1 and has same functions as the sequence represented by SEQ ID No. 1 does. The invention also provides the nucleotide sequence of the protein and a preparation method and application for the same. The protein has good immunogenicity, can effectively prevent infection caused by Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae and Streptococcus uberis, has a wide immune range and can be used for preventing dairy cow mastitis; moreover, while immunoprotection effects of the GIT fusion protein provided in the invention is guaranteed, preparation process and immune procedures in actual production are simplified, the length of the peptide chain of the fusion protein is shortened, expression of the fusion protein is enhanced, and the GIT fusion protein has an important value in development and application of novel vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a GapC for preventing cow mastitis id -IsdB id - Preparation and application of TRAP (hereinafter referred to as GIT) fusion protein, especially a GIT fusion protein capable of preparing vaccines for Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis and Staphylococcus aureus. Background technique [0002] Cow mastitis is an important disease that seriously harms the dairy industry, causing huge economic losses, and has always been a worldwide problem. The etiology of the disease is mainly bacterial infection, among which Staphylococcus aureus (Staphylococcus aureus) and streptococcal infection are the most common, accounting for 55%-80% of the clinical incidence. With the widespread use of antibiotics in the treatment of bacterial infections, a large number of drug-resistant strains have emerged, resulting in little effect of antibiotic treatment, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K39/116A61K39/085A61K39/09A61P31/04
Inventor 崔玉东樊自尧孙虎男朱战波
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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