Recombinant Staphylococcus aureus enterotoxin G oral preparation and use
A technology of Staphylococcus aureus enterotoxin and Staphylococcus enterotoxin, applied in the field of bioengineering, can solve the problems of lack of strict quality control standards, simplicity, and easy introduction of other protein impurities
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Embodiment 1
[0048] Example 1: PCR amplification of the gene sequence encoding the mature peptide of the SEG protein containing restriction sites: design the following primer sequences:
[0049] SEQ ID NO.3: gta caa ccc gat cct aaa tta gac (the underlined part is the restriction site of BamH I)
[0050] SEQ ID NO.4: gga tca gtg agt att aag aaa tac (the underlined part is the Xho I restriction site) uses the genome of Staphylococcus aureus (FRI S6) as a template to amplify the SEG mature peptide gene fragment seg of Staphylococcus aureus enterotoxin;
[0051] The conditions for PCR amplification were as follows. The gene fragments encoding SEG 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
[0056] BSA (5mg / mL): 10μL
[0057] Primer-up (25μmol / L): 4μL
[0058]...
Embodiment 2
[0068] Example 2: Construction of the recombinant plasmid pGEM-T-SEG encoding enterotoxin SEG protein mature peptide gene seg containing restriction sites:
[0069]The gene fragment encoding the mature peptide of SEG protein with restriction sites obtained by PCR amplification was cloned into the pGEM-T plasmid to construct the pGEM-T-SEG recombinant plasmid, which was transformed into Escherichia coli DH5α for amplification. Extract the above recombinant pGEM-T-SEG plasmid, send it for sequencing after enzyme digestion and identification, and the sequencing results show that the gene sequence encoding the mature peptide of the SEG protein with the enzyme digestion site obtained above is 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 G mature peptide has only two more amino acids (Gly-Ser) at the N-termi...
Embodiment 3
[0070] Example 3: Construction of pGEX-4T-1-SEG recombinant expression plasmid containing the gene encoding SEG mature peptide with restriction site:
[0071] The pGEM-T-SEG recombinant plasmid and the pGEX-4T-1 plasmid obtained in Example 2 containing the gene fragment encoding the mature peptide of the SEG protein obtained in Example 2 were respectively digested with BamH I and Xho I. The gene fragment encoding the mature peptide of SEG containing the restriction site and the large fragment of the digestion product of the pGEX-4T-1 plasmid after digestion were respectively recovered and ligated to construct the expression plasmid pGEX-4T-1-SEG. The above 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 result showed that the target gene fragment had been inserted into the vector plasmid. The results of electrophoresis are shown in the attached image 3 , l...
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