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
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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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