Expression and application of truncated sasa protein from Staphylococcus aureus in Escherichia coli
A staphylococcus, golden yellow technology, applied in the field of genetic engineering, can solve the problems of complex extraction and preparation processes
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Embodiment 1
[0038] Embodiment 1, Staphylococcus aureus truncation SasA gene fragment obtains
[0039] According to the reported sequence of Staphylococcus Aureus MASA252 strain (GenBank: BX571856.1), after analyzing the full length of the protein encoded by the gene, design primers to amplify the 142-999bp fragment, and add NdeI and XhoI restriction enzymes to both ends of the tSasA sequence site, and the stop codon was removed, and a sequence encoding 6 histidines was introduced at the 3' end.
Embodiment 2
[0040]Example 2, Staphylococcus aureus truncated SasA expression vector construction
[0041] The amplified tSasA gene was digested with NdeI and XhoI, then ligated into the expression vector pET21a(+) that was also digested with NdeI and XhoI, transformed into Escherichia coli competent cells DH5α, and cultured overnight at 37°C. The next day, single clones were picked and placed in 5 mL LB (Amp+) medium, cultured at 37°C, 220 rpm for 12 hours, plasmids were extracted, NdeI and XhoI double enzymes were digested to identify the insertion of the target gene, and sent for sequencing. The correctly sequenced plasmid was named pET21a-tSasA.
Embodiment 3
[0042] Example 3. Escherichia coli expression and western-blot identification of Staphylococcus aureus truncated SasA
[0043] The pET21a(+) vector correctly connected to the tSasA gene was transformed into Escherichia coli competent cell BL21(DE3), and a single clone was picked and cultured in 5mL LB(Amp+) liquid medium at 37°C and 220rpm until OD600nm≌0.6, and then added IPTG with a final concentration of 1 mM was incubated at 28° C. and 220 rpm for 6 h. Collect the bacteria by centrifugation at 5,000g at 4°C, resuspend in PBS, and then sonicate the bacteria. The supernatant was collected by centrifugation at 12,000g at 4°C, and subjected to SDS-PAGE electrophoresis, and the expression of tSasA protein with a size of 40kDa was identified using the empty vector expression product as a control.
[0044] Western-blot analysis of the specific binding of tSasA protein and mouse anti-His tag monoclonal antibody. SDS-PAGE protein electrophoresis was performed on supernatants of u...
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