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Micrococcus pyogenes alpha-hemolysin and coded sequence thereof

A staphylococcus, golden yellow technology, applied in the field of biotechnology and veterinary medicine, can solve the problems of endangering human health, the spread of mastitis, acute death of dairy cows, etc., and achieve a good antigenic effect

Inactive Publication Date: 2008-05-21
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

Staphylococcus aureus infection destroys the mammary gland tissue, leading to the decline or loss of lactation function of the infected mammary gland. Staphylococcus aureus also releases damaging toxins such as α-hemolysin, etc., which can cause acute death of dairy cows in severe cases; secondly, Staphylococcus aureus is contagious , can often lead to the spread of mastitis in the entire cattle farm; moreover, Staphylococcus aureus is a pathogenic bacterium that can infect humans and animals, can make people sick through the food chain, and seriously endanger human health
[0003] At present, the clinical application of antibiotics to treat and prevent Staphylococcus aureus mastitis has played a certain role, but it has also brought about public health problems such as "resistant milk" and drug-resistant strains that endanger human health. Therefore, this method is receiving more and more attention. 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 alpha-hemolysin and coded sequence thereof
  • Micrococcus pyogenes alpha-hemolysin and coded sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Materials and methods

[0048] 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.

[0049] 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.

[0050] Experimental animals: Kunming mice, weighing 18-22 g, purchased from the Experimental Animal Center of Shandong University of Traditional Chinese Medicine.

Embodiment 2

[0052] PCR amplification of α-HL gene and its sequencing and analysis

[0053] The DNA template of Staphylococcus aureus was extracted by phenol-chloroform method. According to the α-HL gene sequence published on GenBank, the following pair of primers were designed and synthesized:

[0054] Upstream (including BamH I restriction site): 5'-ccgg atcc gcagattctgattattaa-3';

[0055] Downstream (including HindIII restriction site): 5'-tc aagctt atttgtcatttcttctt-3'.

[0056] 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: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, 30 cycles of amplification, and finally extension at 72°C for 5 min.

[0057] The PCR product was connected with the pMD18-T vector, transformed into E.coli DH5α, and the extracted plasmid ...

Embodiment 3

[0059] Construction of recombinant expression plasmids

[0060] Extract the positive plasmid in Example 2, after double digestion with BamH I and HindIII, use the kit to recover the target fragment, and pET32a that has also been double digested with BamH I and HindIII + The expression vector was connected to construct the recombinant expression plasmid pET32a + -α-HL, and carry out BamH I, HindIII double enzyme digestion identification and PCR identification of the recombinant plasmid.

[0061] After double enzyme digestion, there was a specific band at about 879 bp, and a specific band was amplified at about 879 bp through PCR identification, indicating that the recombinant plasmid was constructed correctly (see Figure 2 for identification results).

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Abstract

The invention discloses a golden staphylococcus alpha-hemolysin, a polynucleotide coded as the alpha-hemolysin and a biological method of producing the alpha-hemolysin by the gene engineering technology; at the same time, the invention also discloses the purpose of the golden staphylococcus alpha-hemolysin. The golden staphylococcus alpha-hemolysin of the invention establishes a good foundation for the further research of the pathogenic and immune mechanism and the bacterin of the golden staphylococcus alpha-hemolysin.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and veterinary medicine, in particular, the present invention relates to a novel polynucleotide encoding Staphylococcus aureus α-hemolysin and the polypeptide encoded by the polynucleotide. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. Background technique [0002] Cow mastitis (Mastitis) is the main cause of low production efficiency and high costs 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, leading to the decline or loss of lactation function of the infected mammary gland. Staphy...

Claims

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Application Information

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IPC IPC(8): C07K14/31C12N15/31C07K16/18G01N33/53C12Q1/68
Inventor 杨宏军王长法杨少华高运东仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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