The baculovirus-insect cell system is a binary system consisting of two parts. The first part is the baculovirus expression vector, which is an insect virus whose function is obviously to introduce the foreign gene encoding the target protein into the host cell. Another function of baculovirus expression vectors is to provide the required transcription complex for the transcription of target genes under the control of late or very late promoters. The second part is the host, usually a lepidopteran insect cell line, but sometimes a lepidopteran insect.
In 1983, Smith successfully cloned and expressed human β-interferon under the AcMNPV polyhedron promoter for the first time. At the same time, Hooft et al. reported the nucleotide sequence of the polyhedrin gene. And Pennock confirmed the method of producing recombinant virus, and expressed the polyhedrin-β-galactosidase fusion protein through the sorted recombinant virus. Subsequently, the baculovirus expression vector system (BEVS) began to express the target protein in insects. As the research on the baculovirus promoter becomes more and more thorough, it is found that the baculovirus promoter can express single or multiple foreign proteins alone or in combination with the polyhedrin promoter. This discovery also provided a very important basis for the development of baculovirus expression vectors. In this process, the researchers located the AcMNPV high-efficiency expression gene p10 and determined its sequence, and then constructed AcMNPV that uses the p10 promoter to express foreign proteins. In 1988, Vlak et al. proved that p10 is a non-essential gene, and expressed the β-galactosidase fusion protein with the p10 promoter at the site of p10.
In 1990, Kitts et al. found that linearized baculovirus DNA can improve the efficiency of obtaining recombinant vectors (~25-30%). In 1993, Kitts and Posses inserted an unusual restriction site Bsu36I upstream of the adjacent gene ORF603 and downstream of ORF1629, respectively, of the wild-type baculovirus polyhedrin gene. AcMNPV DNA cut with restriction enzyme Bsu36I can only infect cells after recombination with foreign genes. The recombination rate of the modified baculovirus expression vector and the vector with foreign genes reached 90%, which greatly improved the screening efficiency of recombinant viruses. Subsequently, Luckow and others optimized this system. They modified the AcMNPV DNA and inserted the E. coli mini-F replicon, the kanamycin resistance gene and the bacterial transposon att-Tn7 into the polyhedron gene locus. . The new baculovirus shuttle vector (AcMNPV bacmid) can not only replicate in E. coli like a plasmid, but also infect Lepidopteran insect cells. The recombinant baculovirus DNA is first purified from E. coli and then transfected into insect cells to produce recombinant baculovirus. This is the Bac-to-Bac system. Until 2003, Ian Jones knocked out a partial sequence of ORF1629, so that the baculovirus recombination efficiency reached 100%.
(1) Easy to operate. Baculovirus has a small genome, simple molecular biological characteristics, and a variety of restriction endonuclease sites on the genome.
(2) Large foreign genes can be accommodated. The virion of baculovirus is rod-shaped and has strong plasticity to accommodate foreign genes. In theory, it can accommodate any foreign genes.
(3) High-efficiency expression of foreign genes. In the baculovirus genes, the p10 gene and the polyhedrin gene are both extremely late genes, and their promoters can efficiently express the target protein.
(4) The target protein can be modified after expression. The host of baculovirus is insect cells, and the target protein can be processed and modified using the post-translational modification system of insect cells.
(5) High security. Baculovirus expression vectors are very specific and can only replicate in insect cells.
(6) Low cost. Compared with mammalian cells, insect cultured cell lines are relatively easier to grow, and insect cells can be cultured in suspension to produce large amounts of the target protein.
(1) Use the protein sequence translated from the target gene sequence as the source template, and optimize a set of gene sequences that are more suitable for the SF9 cell line through codons.
(2) According to the results of the target gene sequence analysis, see if the target gene contains a signal peptide. If there is no signal peptide, it is necessary to add the GP67 signal sequence to the N end of the protein sequence to help the protein to be secreted and expressed outside the cell during gene recombination. Adding 6XHis-Tag to the end facilitates the detection and affinity purification of recombinant proteins.
According to the above design ideas, the pFast-bac1-target-gene expression plasmid was constructed by gene synthesis, and the recombinant Bacmid was obtained by the blue-and-white screening method after transformation. After PCR identification, it was transfected into sf9 cells to obtain the P1 generation virus and the P2 generation. Virus. Infect 200ml small test expression, detect the protein expression by western blot, confirm the protein expression, and then obtain the target protein with more than 80% purity by Ni Focurose 6FF (TED) affinity purification.
Medicilon researchers establish a well-developed baculovirus-insect cell expression services platform. We provide the expression and purification services on preparation of recombinant baculovirus and recombinant protein. We have a good track record in producing kinase and recombinant protein complexes.
Generation of recombinant Bacmid DNA
Preparation of recombinant baculovirus
Titration of baculovirus
Protein expression verification and optimization
Small scale expression and purification of recombinant protein in insect cell
Scale up expression and purification of recombinant protein in insect cell
Synthesize the target-gene gene by PAS (PCR-based Accurate Synthesis) method, double-enzyme digestion between BamH I and Hind III connected to the pFast-bac1 vector; transfer the obtained recombinant plasmid into the TOP10 cloned strain, and pick the positive Sequencing of clones. The sequencing spliced sequence is compared with the theoretical sequence, and the result shows that the obtained sequence matches the theoretical sequence 100%, and the following work is started.
2.1 Bacmid strain transformation
(1) Thaw competent cells of DH10Bac E.coli on ice;
(2) Slowly add 200ng of pFastBac1-gene transfer vector to competent cells and mix gently;
(3) Place on ice for 30 minutes, then heat shock at 42°C for 90 seconds;
(4) Quickly stand still on ice for 5 minutes;
(5) Add 900 μl S.O.C. medium to the EP tube;
(6) 37°C, 225 rpm shaking the bacteria for 4h;
(7) Take 100μl of bacterial solution and apply it to LB containing 50 mg/ml kanamycin, 7 mg/ml gentamicin, 10 mg/ml tetracycline, 100 mg/ml x-gal, and 40 mg/ml IPTG flat;
(8) Incubate at 37°C for 48 hours.
2.2 Identification of recombinant Bacmid plasmid
Pick 3 white monoclonal colonies and inoculate them respectively in 5ml containing 50 mg/ml kanamycin, 7 mg/ml gentamicin, 10 mg/ml tetracycline, shaking bacteria, and initial screening of positive clones by PCR. A small amount of plasmids are extracted and PCR is used to verify the correctness of the bacmid. Because the bacmid size is greater than 135Kb, the restriction method is difficult to verify, so PCR is used for verification. Theoretically, if the target gene is successfully transposed into Bacmid, the amplified product The size should be (2300bp + target gene length)
3.1 Cultivation of sf9 cells
sf9 cell culture is cultured with SF 900Ⅱ medium, which is usually sub-flask every 3 days. Sf9 cells with logarithmic growth phase cells and cell viability greater than 95% are used for transfection experiments.
3.2 The method of cryopreservation of Sf9 cells:
Cells are cultured to the logarithmic growth phase and the viability exceeds 90%; count so that the storage concentration is 1×107 /ml to 2×107 /ml; prepare the required amount of storage medium, add DMSO to 10% and FBS to 30% , The pre-cooled culture medium is stored at 4℃; the suspended cells or monolayer cells are centrifuged at 100×g for 5min, and suspended in the pre-cooled cryopreservation medium to the required density; mix well, and dispense into the cryopreservation tube; transfer the cryopreservation tube Put it into a foam box filled with absorbent cotton, and place it at -80°C for 1 day; transfer it to a liquid nitrogen tank for storage.
3.3 Bacmid transfection of sf9 cells
(1) Inoculate 9×105 cells/2ml/well in a six-well plate. The medium Grace’s medium contains penicillin 50U/ml, streptomycin 50ug/ml, and 10% FBS;
(2) Incubate at 27°C for 1 hour to make the cells adhere to the wall;
(3) Prepare the Bacmid and Cellfectin Reagent complex during this period: A. Dilute 1ug Bacmid (about 5ul) with 100ul incomplete Grace’s medium (without double antibody, FBS); B. Invert Cellfectin Reagent 5~10 before use Mix well, take 6ul Cellfectin Reagent and dilute with 100ul incomplete Grace’s medium (without double antibody, FBS); C. Combine the above two diluents (total volume is about 210ul), mix gently, and room temperature Incubate for 15~45min;
(4) During the preparation of the Bacmid and Cellfectin Reagent complex, aspirate the medium in the six-well plate, wash once with 2ml incomplete Grace’s medium (without double antibodies, FBS), and remove the medium;
(5) Add 800ul of incomplete Graces medium (without double antibodies, FBS) into the tube containing 210ul of the complex, mix gently, and add to each well;
(6) Incubate the cells in the six-well plate for 5 hours at 27°C;
(7) Remove the complex mixture, and then add 2ml complete medium (SF900 contains double antibody, 10% FBS) to each well;
(8) Incubate in a humidity incubator at 27°C for 72 hours or until the cells show signs of virus infection. The supernatant and precipitation need to prepare samples separately, and use it for later western blot expression identification.
When the cells show signs of infection, transfer the cell supernatant to a 15ml centrifuge tube and centrifuge at 1000rpm for 5min to remove cells and large debris. It can be filtered with a 0.2um filter with low protein binding rate, and the titer loss is less than 10%; Transfer the virus-containing supernatant to another sterile EP tube with a lid (generally, the titer of P1 is about 10^6pfu/ml), and place the resulting virus liquid in a refrigerator at 4°C (short-term) in the dark. For long-term storage, make 1ml aliquots and store at -80℃ in the dark.
The titer of the primary virus (P1) is low, ranging from 1×106 to 1×107, and the titer after amplification is 1×107 to 1×108. Add an appropriate amount of P1 virus to a 50ml shake flask.
The following formula can be used to calculate when amplifying the virus:
MOI (Multiplicity of infection) is between 0.01 and 0.1,
Inoculation volume (ml): desired MOI (phf/ml) ×(total number of cells) tlter of viral inoculum (puf/ml) The virus collected from infected cells within 48 hours has increased by nearly 100 times, and the virus collected after 48 hours has a higher quality Low. The collection time of each virus has a certain difference, such as collection at 72h, but as the cells are lysed, the proliferation of the virus will be impaired.
There are 1.2 million cells per well in the six-well plate, stand for 1 hour, and the virus is diluted to 101 102 103 104 105 106 107 108. The supernatant in the six-well plate is removed, and the three titers 106, 107, and 108 are added to the In the well plate, parallel control 2 groups, incubate for 1 hour, configure plaque medium, add 2mL plaque medium per well; add neutral red staining solution on the fourth day; count the number of plaques in 7-10 days to calculate the virus titer
sf9 cells were inoculated in a 500ml cell culture flask with a 2×106/ml flask; when the cells grew to logarithmic phase, they were infected with P2 virus to infect sf9 cells; cells and supernatants were collected within 48~72h for detection of recombinant protein expression .
7.1. Scale up cultivation according to the conditions and methods. Centrifuge at 10000g for 20 min at 4℃ and take the supernatant;
7.2. Using a low pressure chromatography system, the supernatant was loaded onto a Ni Focurose 6FF (TED) Binding-Buffer pre-equilibrated Ni Fcourose 6FF (TED) affinity chromatography column at a flow rate of 0.5 ml/min;
7.3. Flush with Ni Focurose 6FF(TED)Binding-Buffer at a flow rate of 0.5 ml/min until the OD280 value of the effluent reaches the baseline;
7.4. Wash with Ni Focurose 6FF(TED) Washing-Buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 ml/min until the OD280 value of the effluent reaches the baseline;
7.55. Use Ni Focurose 6FF (TED) Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) to elute the target protein at a flow rate of 1 ml/min, and collect the effluent;
7.6. Add the protein solution collected above into a dialysis bag, and use PBS (pH 7.4) for dialysis overnight;
7.7. Perform 10% SDS-PAGE analysis,
8.1. The sample is loaded with 0.01ug.
8.2. After loading the sample, run the polyacrylamide gel at 90V to finish the stacking gel, and then increase the voltage to 200V until the end of the electrophoresis.
8.3. After the electrophoresis is over, remove the gel and transfer the membrane, and transfer the membrane at a constant voltage of 100V for about 1.5 hours.
8.4. After the electroporation, remove the membrane and wash with PBS 4 times, 5 minutes each time. Then place it in 5% skimmed milk powder blocking solution and block at 37°C for 1 hour.
8.5. Dilute the primary antibody with blocking solution, and react the membrane in the primary antibody diluent at 37°C for 1 hour.
8.6. Wash the membrane 4 times, 5 minutes each time; dilute the secondary antibody with a blocking solution containing 5% milk. The membrane was reacted in the secondary antibody at 37°C for 1 hour.
8.7. Wash film ECL development.
Introduction of Bac-to-Bac System
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