Recombined staphylococcus aureus enterotoxin M oral preparation and application thereof
A technology of Staphylococcus aureus enterotoxin and staphylococcus entero, applied in the field of bioengineering, can solve the problems of simplicity, instability between batches, and easy introduction of other protein impurities.
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Embodiment 1
[0046] Example 1: PCR amplification of the gene sequence encoding the mature peptide of the SEM protein containing restriction sites:
[0047] Design the following primer sequences:
[0048] SEQ ID NO. 3: ca g gat cc t ttt gct att cgc aaa atc ata tcg ca (the underlined part is the BamHI restriction site)
[0049] SEQ ID NO.4: gc c tcg ag t caa ctt tcg tcc tta taa gat att tct ac (the underlined part is the XhoI restriction site)
[0050] Using the genome of Staphylococcus aureus (FRI 1230) as a template, it is used to amplify the mature peptide gene fragment sem of Staphylococcus aureus enterotoxin SEM;
[0051] The conditions for PCR amplification were as follows. The gene fragments encoding SEM mature peptides were amplified, wherein each gene fragment had a BamH I restriction site at the 5' end and an Xho I restriction site after the terminator at the 3' end.
[0052] PCR system:
[0053] h 2 O: 60 μL
[0054] Buffer(10×): 10μL
[0055] Mg 2+ (25mmol / L): 8μL
[...
Embodiment 2
[0068] Example 2: Construction of the recombinant plasmid pGEM-T-SEM encoding enterotoxin SEM protein mature peptide gene sem containing restriction sites:
[0069]The gene fragment encoding the mature peptide of SEM protein with restriction sites obtained by PCR amplification was cloned into the pGEM-T plasmid to construct the pGEM-T-SEM recombinant plasmid, which was transformed into Escherichia coli DH5α for amplification. The above recombinant pGEM-T-SEM plasmid was extracted, identified by enzyme digestion, and then sent for sequencing. The sequencing results showed that the gene sequence encoding the mature peptide of the SEM protein with the enzyme digestion site obtained above was different from the corresponding gene sequence on GenBank except at the 5' The rest of the site is exactly the same except for the addition of a restriction site, and the amino acid sequence of the corresponding enterotoxin M mature peptide has only two more amino acids (Gly-Ser) at the N-term...
Embodiment 3
[0070] Example 3: Construction of pGEX-4T-1-SEM recombinant expression plasmid containing the gene encoding SEM mature peptide with restriction site:
[0071] The pGEM-T-SEM recombinant plasmid and the pGEX-4T-1 plasmid obtained in Example 2 containing the gene fragment encoding the mature peptide of the SEM protein obtained in Example 2 were respectively digested with BamH I and Xho I. The gene fragment encoding the mature peptide of SEM containing the restriction site and the large fragment of the pGEX-4T-1 plasmid digestion product after digestion were respectively recovered and ligated to construct the expression plasmid pGEX-4T-1-SEM. The above-mentioned recombinant expression plasmid was transformed into Escherichia coli DH5α to amplify and extract the plasmid. After digestion and identification by BamH I and Xho I, the results showed that the target gene fragment had been inserted into the vector plasmid. The electrophoresis results are shown in Figure 3 of the specifica...
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