Pseudomonas aeruginosa vaccine recombinant protein SBP, and preparation method and applications thereof
A Pseudomonas aeruginosa, recombinant protein technology, applied in recombinant DNA technology, chemical instruments and methods, antibacterial drugs, etc. problem, to achieve good anti-PA infection, good protection effect, easy separation and purification effect
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[0036] Such as figure 1 As shown, the preparation method of the Pseudomonas aeruginosa vaccine recombinant protein SBP provided by the embodiments of the present invention comprises the following steps:
[0037] S101: Take 400 μL of the spare pGEX-6p-1-SBP / XL-1blue bacterial solution stored in a refrigerator at 4°C and add it to 20 mL of LB medium containing Amp resistance for one activation. After culturing at 200 rpm and 37°C for 5-6 hours, Take 10mL of the primary activated bacterial liquid and add it to 1000mL LB medium containing Amp resistance for secondary activation, and culture at 37°C for 3-4h until OD600 is 1.0;
[0038] S102: After adding 200 μL IPTG (final concentration of 200 μM) and placing it in a shaker at 16° C. for overnight induction, centrifuge at 12,000 rpm for 15 minutes to collect the bacterial cells, add 50 mL of lysis buffer (same as Example 2) to resuspend the bacterial cells, and then ultrasonicate the bacterial liquid Lyse for 3min (200V), collect...
Embodiment 1
[0051] Embodiment 1: The cloning of SBP gene and the construction of recombinant plasmid pGEX-6P-1-SBP
[0052] 1. Firstly, according to the full-length gene sequence of the SBP protein of Pseudomonas aeruginosa PA01, bioinformatics software was used for structural analysis to determine the SBP target gene fragment to be amplified.
[0053] 2. According to the analysis results, the PCR method was used to amplify the SBP target gene fragment from the PA01 genome, and the amplification steps were as follows:
[0054] 1) Design the PCR primers as follows, respectively SEQ ID NO: 3-4 (the base sequence of the restriction site is underlined)
[0055]
[0056] 2) Take out the preserved Pseudomonas aeruginosa strain PA01 from the freezer at -80°C and spread it on LB solid medium, culture it at 37°C overnight, then pick a single colony and inoculate it in LB liquid medium for 8 hours , referring to the bacterial genome extraction kit to extract the PA01 genome.
[0057] 3) Using ...
Embodiment 2
[0084] Example 2: Induced expression, purification and identification of expression form of recombinant fusion protein SBP in prokaryotic expression system-Escherichia coli
[0085] 1. SBP induced expression
[0086] 1) Take 100 μL of the overnight cultured pGEX-6p-1-SBP / XL-1blue bacterial solution and add it to 10 mL of Amp+ resistant LB medium, culture overnight at 180 rpm at 37°C, take 400 μL of the overnight cultured bacterial solution and add 20 mL of Amp+ resistant LB medium (the rest of the bacterial solution was stored in a 4°C refrigerator for later use), cultured at 37°C for 2-3 hours at a rotation speed of 200 rpm, and when the secondary activation reached OD600 of 0.6-0.8, 4 μL (1mol / L) of IPTG was added to make The final concentration was 200 μM, and then placed on a shaker to induce expression at 30° C. for 3 hours.
[0087]2) Take out the bacterial solution after induced expression, centrifuge at 12000rpm for 5min, discard the supernatant, add 1mL of lysisbuffe...
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