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Micrococcus pyogenes adhesion functional peptide and coded sequence thereof

A staphylococcus, golden yellow technology, applied in the direction of peptides, depsipeptides, antibacterial drugs, etc., can solve problems such as danger and limitation

Inactive Publication Date: 2008-05-14
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the application of antibiotics to prevent and treat Staphylococcus aureus mastitis has achieved a certain clinical effect, but it has also brought about public health problems such as "resistant milk" and drug-resistant strains that endanger human health. Therefore, it is subject to more and more restrictions.
Vaccination prevention is the most convenient, effective and economical measure, but there is no staphylococcus aureus vaccine with independent intellectual property rights available for production in China

Method used

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  • Micrococcus pyogenes adhesion functional peptide and coded sequence thereof
  • Micrococcus pyogenes adhesion functional peptide and coded sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Materials and methods

[0049] Strains and plasmids: The clinical isolates of Staphylococcus aureus cow mastitis were isolated and identified in a conventional manner; Escherichia coli BL21(DE3); pMD18-T vector was purchased from TaKaRa; pET32a + Vectors were purchased from Novagen.

[0050] Reagents: high-fidelity DNA polymerase, restriction endonucleases (BamH I and HindIII), T 4 DNA ligase was purchased from TaKaRa; DNA gel recovery kit was purchased from U-gene; ampicillin was purchased from Huamei Company; Ni-NTA affinity chromatography column was a product of QIAGEN.

[0051] Experimental animals: Kunming mice, weighing 18-22 g, purchased from the Experimental Animal Center of Shandong University of Traditional Chinese Medicine.

Embodiment 2

[0053] PCR amplification of Fnbp gene and its sequencing analysis

[0054] The DNA template of Staphylococcus aureus was extracted by phenol-chloroform method. According to the Fnbp gene sequence published on GenBank, a pair of primers were designed and synthesized:

[0055] Upstream (including BamH I restriction site): KF1: 5′-AAA GGATCC GCATCAGAACAAAAGACA-3';

[0056] Downstream (including Sal I restriction site): KF2: 5′-AGA GTC GAC CTACTCAGAGGACTCAGTGTA-3'.

[0057] 50μL PCR reaction system: 5μL 10×PCR buffer, Mg 2+ (25mM) 3μL, dNTP (25mM) 4μL, Taq enzyme (2.5U) 0.5μL, each primer 1μL, template 2μL, sterile double distilled water 33.5μL. Cycle conditions: 94°C pre-denaturation for 5 minutes; 94°C for 1 minute, 50°C for 1 minute, 72°C for 3 minutes, 30 cycles; 72°C for 10 minutes.

[0058] The PCR product was connected with the pMD18-T vector, transformed into E.coli DH5α, and the extracted plasmid was identified by agarose electrophoresis after double digestion with...

Embodiment 3

[0060] Construction of recombinant expression plasmids

[0061] Extract the positive plasmid in Example 2, after double digestion with BamH I and Sal I, use the kit to recover the target fragment, and pET-32a that has also been double digested with BamH I and Sal I + The expression vector was connected to construct the recombinant expression plasmid pET32a + -Fnbp, and carry out BamH I, Sal I double enzyme digestion identification and PCR identification on the recombinant plasmid. After double enzyme digestion, there was a specific band at about 1.7kb, and a specific band was amplified at about 1.7kb through PCR identification, indicating that the recombinant plasmid was constructed correctly (see Figure 2 and 3 for the identification results).

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Abstract

The invention discloses a golden staphylococcus adhesin functional peptide for milk cow mastitis, a polynucleotide for coding adhesin protein and a biological method for preparing adhesin protein function by the genetic engineering; simulantesouly, the invention discloses the possible uses of the golden staphylococcus adhesin functional peptide in related field. The invention has the advantages of helping to further research the pathogenesis of the mastitis caused by the golden staphylococcus and the corresponding vaccine.

Description

technical field [0001] The invention belongs to the field of veterinary medicine, in particular, the invention relates to a new polynucleotide encoding bovine Staphylococcus aureus adhesin functional peptide and a polypeptide encoded by the polynucleotide. The present invention also relates to the possible use and preparation of this polynucleotide and functional polypeptide. Background technique [0002] Dairy cow mastitis (Mastitis) is the main cause of low production efficiency and high cost in the dairy industry. The annual loss of dairy cows due to mastitis in the country reaches more than 10 billion yuan. At present, it has been proved that there are more than 150 kinds of microorganisms that cause mastitis in dairy cows, among which the mastitis caused by Staphylococcus aureus infection accounts for about 30.6%-41.7%. Staphylococcus aureus infection destroys the mammary gland tissue, resulting in the decline or loss of lactation function of the infected mammary gland...

Claims

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Application Information

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IPC IPC(8): C07K14/31A61K39/085A61K39/395A61P31/04G01N33/68
Inventor 杨宏军杨少华王长法高运东仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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