Method for purifying pseudomonas aeruginosa vaccine recombinant protein Vac 9
A Pseudomonas aeruginosa purification method technology, applied in the field of biopharmaceuticals, can solve the problems of recombinant protein Vac9 purification methods and reports that have not yet been seen, and achieve good immune protection, good repeatability, and high humoral immune response.
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[0064] Example 1: Cloning of OprL gene and construction of recombinant plasmid pGEX-6P-2-OprL
[0065] 1. First, according to the full-length gene sequence of the OprL protein of Pseudomonas aeruginosa PA01, use bioinformatics software to conduct structural analysis to determine the OprL target gene fragment that needs to be amplified.
[0066] 2. According to the analysis results, the PCR method was used to amplify the OprL target gene fragment from the PA01 genome. The amplification steps are as follows:
[0067] 1) Design PCR primers as follows, respectively SEQIDNO: 3-4 (the base sequence of the restriction site is underlined)
[0068] Seq ID
[0069] 2) Pseudomonas aeruginosa strain PA01 taken out of the freezer at -80°C was spread on LB solid medium, cultured overnight at 37°C, and then a single colony was picked and inoculated in LB liquid medium for 8 hours , Refer to the bacterial genome extraction kit to extract the PA01 genome.
[0070] 3) PCR amplification of OprL gene f...
Example Embodiment
[0098] Example 2: Induced expression of recombinant fusion protein Vac9 in a prokaryotic expression system-Escherichia coli and identification of the expression form
[0099] 1. Induced expression of recombinant protein Vac9
[0100] Take 100μL of the pGEX-6P-2-OprL / XL-1blue cultured overnight and add it to 10mL Amp+ resistant LB medium, culture overnight at 180rpm at 37℃, respectively take 400μL of the overnight cultured bacterial solution and add 20mL Amp+ resistant LB medium (The remaining bacterial solution is stored in a refrigerator at 4°C for later use), cultured at 37°C for 2 to 3 hours, at a rotation speed of 200 rpm, and after the second activation to an OD600 of 0.8-1.0, add 4 μL of IPTG to make the final concentration 200 μM, and then place it on a shaker Induced expression at 30℃ for 3h.
[0101] 2) Take out the bacterial solution after induced expression, centrifuge at 12000rpm for 5min, discard the supernatant, add 1mL lysisbuffer (20mMPB, pH7.2, 250mMNacl) and mix we...
Example Embodiment
[0108] Example 3: Preparation of recombinant protein Vac9 antigen
[0109] 1. Amplify the culture to obtain protein
[0110] Take 400μL of pGEX-6P-2-OprL / XL-1blue bacterial solution stored in the refrigerator at 4℃ for later use and add it to 20mL of Amp-resistant LB medium for one activation. After incubating at 200rpm at 37℃ for 5-6 hours, take 8mL once The activated bacteria solution was added to 400 mL of Amp-resistant LB medium for secondary activation. After culturing at 37°C for 3 to 4 hours until the OD600 reached 1.0, add 80 μL of IPTG (final concentration of 200 μM) and place in a 16°C shaker overnight for induction. Afterwards, the cells were collected by centrifugation at 12000rpm for 15min, and 20mL of lysisbuffer (same as in Example 2) was added to resuspend the cells. The bacteria solution was subjected to ultrasonic lysis for 3min (200V). Company) Gel beads (beads) binding treatment; then SDS-PAGE gel electrophoresis.
[0111] 2. Use restriction digestion method to ...
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