Pseudomonas aeruginosa vaccine recombinant protein rePilA-FliC and preparation method and application
A technology of Pseudomonas aeruginosa and recombinant protein, applied in the direction of recombinant DNA technology, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems that are difficult to meet the application of vaccine antigens, difficult to prepare FliC, easy to aggregate and degrade, etc. problem, to achieve good protection effect, good PA infection, and low production cost
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Embodiment 1
[0033] Example 1 Synthesis and subcloning of genes
[0034] rePilA-FliC protein is composed of PilA protein (Ala35-Arg150) and FliC protein (Asn23-Ala401) connected by flexible Linker-GSGGSG molecular fusion. Introduce BamHI restriction sequence at the 5' end of its coding sequence, introduce TGA stop codon and Xhol restriction sequence at its 3', connect to vector pGEX-6p-2 through BamHI and Xhol restriction sites, and obtain recombinant plasmid pGEX - The synthesis of the DNA sequence (SEQ ID NO: 2) of 6p-reSBP-FliC and rePilA-FliC and the connection of the sequence and pGEX-6p-2 were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0035] The amino acid sequence of rePilA-FliC is shown in SEQ ID NO:2.
[0036] AlaArgSerGluGlyAlaSerAlaLeuAlaSerValAsnProLeuLysThrThrValGluGluAlaLeuSerArgGlyTrpSerValLysSerGlyThrGlyThrGluAspAlaThrLysLysGluValProLeuGlyValAlaAlaAspAlaAsnLysLeuGlyThrIleAlaLeuLysProAspProAlaAspGlyThrAlaAspIleThrLeuThrPheThrMetGlyGlyAlaGlyProLysAsnLysGlyLys...
Embodiment 2
[0045] Example 2 Transformation of recombinant plasmid and identification of double enzyme digestion
[0046] 1. Transformation of recombinant plasmids Take 3 tubes of Escherichia coli XL1 blue competent cells from the -80°C refrigerator, and add 1ulpGEX-6p-rePilA-FliC to synthesize the plasmid. Ice bath for 10min, heat shock in 42℃ metal bath for 90s, rapid ice bath for 2min. Add 600 μl LB blank medium, mix well, and shake at 220 rpm for 1 h in a shaker at 37°C. Centrifuge at 5000 rpm for 3 min at room temperature, discard 300 μl of supernatant, resuspend the bacteria, take 200 μl and spread on Amp-resistant LB plate. Plates were placed upside down in a 37°C incubator for 24 hours.
[0047] Pick well-separated colonies on the transformation plate, inoculate them in Amp-resistant LB medium, and culture them overnight at 37°C with shaking.
[0048] 2. Identification by double enzyme digestion
[0049] Take the positive plasmid cultured overnight on a shaker at 37°C, and ext...
Embodiment 3
[0052] Example 3 Recombinant fusion protein rePilA-FliC induced expression, purification and expression form identification in prokaryotic expression system-Escherichia coli
[0053] 1. Induced expression of rePilA-FliC
[0054] 1) Take 100 μL of the overnight cultured pGEX-6P-2-rePilA-FliC / 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+ In the resistant LB medium (the rest of the bacterial solution is stored in a 4°C refrigerator for later use), incubate at 37°C for 2-3 hours at a rotation speed of 200rpm. to 200 μM, and then placed on a shaker to induce expression at 30°C for 3 hours.
[0055] 2) Take out the bacterial solution after induced expression, centrifuge at 12000rpm for 5min, discard the supernatant, add 1mL lysisbuffer (20mM PB, pH 7.2, 250mMNacl) to mix, ultrasonically lyse for 3min (sonication 6 times 30s / time), and t...
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