A method for expressing fsa1, a cat flea flea allergen in saliva
The technology of flea saliva and FSA1 is applied in the field of bioengineering, which can solve the problems of dependence on flea extracts and high cost, and achieve the effects of reducing production cost, improving production efficiency and simple operation.
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Embodiment 1
[0037] Embodiment 1: Construction of recombinant FSA1 expression plasmid
[0038] Use artificially synthesized FSA1 as a template, and use SEQ ID No.1 and SEQ ID No.2 as specific primers for PCR amplification; take 5 μL of the reaction product for electrophoresis on 1% agarose gel, and gel imaging system to detect the amplification result , the amplification result is as figure 1 shown.
[0039] SEQ ID No.1: 5'-CGGGAATTCATGGAAGATATTGG-3';
[0040] SEQ ID No.2: 5'-CGGCTCGAGTTATTTTATTTTTTGG-3';
[0041] PCR amplification system 25 μL: Exteq enzyme 12.5 μL, DNA template 1 μL, upstream primer and downstream primer 0.5 μL each, add ddH 2 0 to 25 μL;
[0042] The PCR amplification reaction program was: 94°C pre-denaturation for 5 minutes, 94°C for 30s, 53°C for 30s, 72°C for 30s, a total of 35 cycles, and finally 72°C for 10 minutes.
[0043] After purifying the obtained PCR amplification product with a gel recovery and purification kit, the amplified product was double-dige...
Embodiment 2
[0044] Example 2: Induced expression and crude purification of FSA1 prokaryotic expression protein
[0045]Streak-inoculate the positive recombinant Escherichia coli obtained in Example 1 on a kana-resistant LB plate, culture overnight at 37°C, pick a single colony and inoculate it in 10mL LB culture medium the next day, culture it with shaking at 200rpm, and cultivate it until the OD600 reaches 0.6. Add IPTG to a final concentration of 0.1mmol / L to induce expression. After inducing expression for 4 hours, ultrasonically disrupt the induced expression bacteria solution, and centrifuge the induced expression bacterial solution after ultrasonic disruption to collect inclusion bodies. The inclusion body was washed, and the washing solution was an aqueous solution containing final concentrations of 50 mM Tris-HCl, 1 mM EDTA, 1% Triton-X100 by volume, and 100 mM NaCl, and the pH of the inclusion body washing solution was 8.0. After washing, centrifuge at 8000rpm / min for 10min an...
Embodiment 3
[0046] Example 3: Fine purification of FSA1 prokaryotic expression protein Ni-NTA column affinity chromatography
[0047] Use 5 times the column volume of Buffer A to equilibrate the affinity chromatography column, purify the soluble protein obtained in Example 2 through Ni-NTA column affinity chromatography to adsorb the target protein, and then use 5 times the column volume of Buffer A to equilibrate the chromatography column , then use 10 times the column volume Buffer A+B to elute the impurity protein, and finally use Buffer B to elute and collect the target protein. The collected target protein solution is detected by the protein chromogenic solution as nearly colorless to confirm that the collection is complete. Buffer A contains KCl with a final concentration of 300mM and KH with a final concentration of 50mM 2 PO 4 , an aqueous solution of urea with a final concentration of 5-10M; Buffer B contains KCl with a final concentration of 300mM and KH with a final concentr...
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