Recombinant Protein Baculovirus Expression Services - Protocol
1 Construction of expression vectors
1.1 Codon optimization
Codon optimization
1.2 Gene synthesis and vector construction
X1xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxX2
The gene is synthesized with enzymatic cleavage sites, X1,X2 respectively, and cloned into the pFastBac vector.
1.3 Plasmid identification
The target gene was inserted into the pFastBac vector. After the bacterial solution was identified correctly by PCR or enzyme digestion, it was sent for sequencing.
2 Construction and transformation of recombinant Bacmid
- E. coli DH10Bac receptor cells were thawed on ice; and
- Transform the correctly sequenced pFastBac1 plasmid into DH10 Bac receptor cells.
- Add 900 ul of LB medium to the EP tube and incubate at 37℃, 220 r/min for 4h.
- Take 100 ul of bacterial solution and apply it on LB plate containing 50ug/ml kanamycin, 7ug/ml gentamicin, 7ug/ml tetracycline, 40ug/ml X-gal, 40ug/ml IPTG
- Invert the plate to 37℃ constant temperature incubator for 48h, 37℃ overnight.
3 Identification of recombinant Bacmid plasmids
- Pick white monoclonal colonies were inoculated in 4 ml of LB medium respectively.
- Incubate at 37℃, 220r/min overnight.
- Extract the recombinant Bacmid plasmid with baculovirus shuttle vector Bacmid small volume extraction kit and sequence.
4 Transfection and harvesting of viruses
- Cultivate Sf9 insect cells with medium at 27℃ without CO2 until the cell density reaches 70%-90%.
- Cultivate the cells at 27℃ for 1h to make the cells stick to the wall.
- Transfect Bacmid plasmid into Sf9 insect cells with insect transfection reagent, continue to culture and observe the cell morphology.
- Incubate at 27℃ until the cells are infected with virus; when all the cells are lysed, collect the supernatant of cell culture fluid, which is the P1 generation recombinant baculovirus.
5 P2 generation virus amplification and purification validation
5.1 P2 generation virus amplification
- Take 20 mL of SF9 cells with a density of 2.0x106/mL and inoculate them into a 250mL trivet;
- P1 generation virus infected Sf9 cells (2*106/ml) that were adhered to the wall for about 1h with a viral infection complex of 0.1;
- Incubate at 27 ℃, 120 rpm for 72 h. Collect the culture medium and centrifuge at 500xg for 5 min, the supernatant obtained is P2 generation baculovirus;
5.2 Purification
- Concentrate the supernatant containing recombinant protein by filtration in an ultrafiltration tube, discard the effluent and save the retention. Transfer the supernatant to another sterile 50mL tube;
- Store the supernatant as P2 virus at 4°C, protected from light;
- The precipitate was resuspended in buffer (PBS, pH 6.0), ultrasonically broken and centrifuged, and the supernatant was purified by Ni column using SDS-PAGE and Western Blot.
6 Experimental results
6.1 Identification of Bacmid plasmids
Figure 1 PCR validation: lanes 1-4 are strongly positive monoclonal, fragment size is consistent with expected theoretical size
6.2 P2 generation test results
Figure 2 SDS-PAGE results of xx protein expression. m: Marker; lane 1: flow-through; lane 2: protein purified sample
Figure 3 WB results of xx protein end product M: Marker; lane 1: purified protein product
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