Establishment of an insect baculovirus expression system
Establishment of an insect baculovirus expression system
The baculoviruses are double-stranded DNA viruses that infect mainly insects, but also in vitro insect cell lines, and are not toxic to humans or animals. Alfalfa silver-striped night moth nucleopolyhedrovirus (AcMNPV) is currently the most widely hosted and used baculovirus.
It has established a widely used Bac-to-Bac baculovirus expression system with the grass-covetous nightshade moth Sf21 cell line, which expresses exogenous genes, with a high level of expression, and is capable of product processing, modification, and folding to form biologically active products.
It is one of the four major expression systems in the field of genetic engineering today and has expressed thousands of exogenous genes.
Principle
The technical principle of Bac-to-Bac system is that the system mainly includes donor plasmid pFastBac, E. coli DH5a, DH10Bac, and insect Sf21 cells.
The donor plasmid pFastBac has the sequence of the left and right arms of Tn7, and between the two arms is the strong promoter of the viral polyhedrin gene polyhedrin and its downstream polyclonal site, polyA site, gentamicin resistance gene and so on.
The exogenous gene was ligated to the donor plasmid, transformed DH5a receptor cells, extracted the recombinant donor plasmid, and then transformed DH10Bac receptor cells, which contained the shuttle plasmid Bacmid and auxiliary plasmid helper, with the F replicon, kanamycin resistance gene, and the gene coding for the peptide segment of lacZa on the Bacmid, and the bacterial transposon Tn7 on the LacZa gene attachment site mini-attTn7.
Tn7 of the recombinant donor plasmid was transposed to the mini-attTn7 site on the Bacmid with the help of the helper-encoded transposase in DH10Bac, which interfered with the expression of LacZ, so that DH10Bac with a recombinant Bacmid was resistant to gentamicin, kanamycin, tetracycline (the helper plasmid was resistant to tetracycline), and to X-gal containing, IPTG to form white colonies on culture plates.
Recombinant Bacmid was extracted and liposomal method was used to transfect insect Sf21 cells, and the recombinant virus could be obtained in the culture medium, the recombinant virus could continue to infect the insect cells, and the exogenous genes were expressed with the virus amplification.
The donor plasmid pFastBacDUAL has two cloning sites, one located downstream of the promoter of poly-hedrin and one located downstream of the p10 promoter.
The recombinant virus constructed with it expresses two proteins after infecting the cells, and some scholars constructed the green fluorescent protein gene downstream of the p10 promoter, and the green fluorescence expressed serves as a marker protein.
Operation method
Establishment of a baculovirus expression system
Principle
The technical principle of Bac-to-Bac system is that the system mainly includes donor plasmid pFastBac, E. coli DH5a, DH10Bac, insect Sf21 cells and so on. The donor plasmid pFastBac has the left and right arms of Tn7, and between the two arms is the strong promoter of the viral polyhedrin gene polyhedrin and its downstream polyclonal site, polyA site, gentamicin resistance gene, etc. The exogenous gene is connected to the donor plasmid pFastBac, and then the exogenous gene is connected to the donor plasmid pFastBac. The exogenous gene was ligated to the donor plasmid, DH5a receptor cells were transformed, the recombinant donor plasmid was extracted, and then DH10Bac receptor cells were transformed. The cells contained the shuttle plasmid Bacmid and helper plasmid, which contained the F replicon, the kanamycin resistance gene and the gene encoding the lacZa peptide, and the mini-attTn7 attachment site of the bacterial transposon Tn7 on the LacZa gene. Tn7 of the recombinant donor plasmid was transposed to the Bacmid with the help of transposase encoded by the helper in DH10Bac. The recombinant donor plasmid Tn7, with the help of the transposase encoded by the helper in DH10Bac, was transposed to the mini-attTn7 site on the Bacmid, which interfered with LacZ expression. As a result, DH10Bac with recombinant Bacmid formed white colonies on plates containing gentamicin, kanamycin, tetracycline (the helper plasmid is resistant to tetracycline), and X-gal and IPTG. Recombinant Bacmid is extracted and liposome method is used to transfect insect Sf21 cells, and recombinant virus can be obtained in the culture medium. Recombinant virus can continue to infect insect cells and exogenous genes are expressed with virus amplification. The donor plasmid pFastBacDUAL has two cloning sites, one is located downstream of the promoter of poly-hedrin and the other is located downstream of the p10 promoter, and the recombinant virus constructed with it expresses two kinds of proteins after infecting the cells. Some scholars constructed the green fluorescent protein gene downstream of the p10 promoter, and the green fluorescence expressed was used as a marker protein.
Materials and Instruments
Equipment: Move The basic process of establishing an insect baculovirus expression system can be divided into the following steps: (I) Reagent preparation (1) LB liquid medium: 10 g of NaCl, 5 g of yeast extract, 10 g of peptone, then adjust the pH to 7.0 with NaOH, volume up to 1000 mL, and sterilize at 103.4 kPa for 20 min. LB solid medium: add 15 g of agar powder per liter of LB liquid medium, and sterilize for 20 min. (2) Luria Agar solid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar powder per liter, pH 7.0~7.2, 15 lbf/in2, sterilized at 103.4 kPa for 20 min. (3) SOC medium: Prepare SOB medium: tryptone 2 g, yeast extract 0.5 g, NaCl 0.05 g, 250 mmol/L KCl solution 1 mL, and then adjust the pH to 7.0 with NaOH, sterilize at 10 lbf/in2, 68.9 kPa for 30 min, and place at 4 ℃. To 10 mL of SOB medium, add 50 μL of sterilized 2 mol/L MgCl2 solution and 200 μL of 1 mol/L glucose in sterile filtration. (4) Grace's complete culture medium: Grace's insect cell culture medium was supplemented with sodium bicarbonate 0.35 g/L, lactalbuminhydrolysalate and yeastolate 3.33 g/L each, then fully dissolved in ultrapure water, adjusted the pH to 6.0 with 1 mol/L NaOH, and then fixed to 1 L. The medium was then filtered aseptically and partitioned and stored at -20℃. 20 ℃. Add fetal bovine serum to 10% when used. (II) Recombinant donor plasmid construction (1) Ligation: Select restriction endonucleases such as EcoRI/XhoI to digest the donor plasmid and exogenous gene fragments respectively, and then ligate with T4 ligase at 16 ℃ overnight. (2) Transfection: 5 μL of DH5a ligand was added to 100 μL of melted DH5a cells in an ice bath for 30 min, heat-excited at 42 ℃ for 60 s, cooled on ice for 2 min, added 400 μL of SOC medium, incubated at 37 ℃, 225 r/min for 1 h, and then centrifuged to collect the cells. The cells were collected by centrifugation. The cells were diluted with appropriate amount of SOC medium and spread on LB solid medium plate containing ampicillin 100 μg/mL, X-gal 100 μg/mL and IPTG 40 μg/mL, and cultured in inverted position at 37 ℃ for 24~48 hours. (3) Screening of blue and white spots: Pick up several independent white spots in the center of the plate, PCR and enzyme digestion to verify whether the exogenous gene has been recombined. (4) Recombinant plasmid extraction: positive white spots were amplified and cultured, and the recombinant donor plasmid was extracted by plasmid extraction kit. (iii) Transposition to obtain recombinant bacmid (1) Transformation: DH10Bac 1 μL of recombinant donor plasmid was diluted into 10 μL of water and added into 100 μL of melted DH10Bac receptor cells in an ice bath for 30 min, heat-excited at 42 ℃ for 45 s, and then cooled on the ice for 2 min. 900 μL of SOC medium was added, and cultured in 37 ℃ at 225 r/min for 4 h. Cells were centrifuged and diluted in SOC medium. The cells were collected by centrifugation and diluted with appropriate amount of SOC medium. The cells were collected by centrifugation and diluted with appropriate amount of SOC medium. The cells were spread on Luria solid medium plates containing kanamycin 50 μg/mL, gentamicin 7 μg/mL, tetracycline 10 μg/mL, X-gal 100 μg/mL and IPTG 40 μg/mL, and then cultured in inverted position at 37 ℃ for 24-48 hours. (2) Screening of blue and white spots: Pick up several independent white spots in the center of the plate, and PCR and enzyme digestion to verify whether the exogenous gene has been recombined. (3) Recombinant plasmid extraction: amplify and culture the positive white spots, and extract the recombinant bacmid with the plasmid extraction kit. pFastBacDUAL with GFP-tagged protein. (iv) Transfection (1) Add 10 μL of cellfectin to 25 μL of sterile water and mix gently for 5 min. (2) Add 25 μL of recombinant Bacmid (control group was replaced by Bacmid without exogenous gene) to liposomes in a water bath at 60 ℃ for 1 h (to inactivate the microorganisms) and mix gently at the bottom of the tubes every 10 min for 1 h at room temperature. (3) Replace the serum-containing culture medium of Sf21 cells in the exponential growth phase with 2 mL of serum-free Grace's medium, and then replace it with 2 mL of new serum-free Grace's medium after 1 h. The above Bacmid/liposome was added to the liposomes at room temperature for 1 h. The above Bacmid/liposome mixture was gently dispensed onto the culture medium and incubated at 27 ℃ for 5~6 h. After that, the culture medium was changed to 2 mL of Grace's complete culture medium containing 50 U/mL Gentamicin and 10% fetal bovine serum. (4) Continue to incubate at 27 ℃ for 3~5 d, and then collect the supernatant culture medium and store it at 4 ℃ to avoid light for infection. (E) Amplification by infection and collection of recombinant proteins (1) When Sf21 cells grow to 80% density, discard the culture medium and add the supernatant culture medium containing the recombinant virus collected after transfection as described above. (2) After static incubation at 27 ℃ for 1.5 h, replace 4 mL of new Grace culture medium and continue to incubate for 3~5 d. Collect the supernatant (containing Budded virus (BV)) and the cells respectively, and carry out the purification and identification of recombinant expression products. (3) The supernatant containing recombinant virus Budded virus can be centrifuged to discard the cell debris and continue to be used for infection. (F) Detection of recombinant proteins by SDS-page or Western blotting method Caveat (1) The PCR product of the amplified exogenous gene should have a restriction endonuclease site sequence at both ends that matches the restriction endonuclease sequence of the donor plasmid to be ligated.(2) Antibiotics, X-gal and IPTG should be spread evenly on plates of transformed DH5a and DH10Bac solid medium and placed in an incubator for half an hour before use, otherwise they will be too toxic to the cells of the organism to form colonies.(3) Recombinant Bacmid should be inactivated by 60 ℃ water bath before mixing with liposomes to avoid contamination during transfection culture. Before transfection, Sf21 cells should be replaced with serum-free Grace's medium, and 2 mL of new serum-free Grace's medium should be replaced after 1 h before adding Bacmid/liposome mixture, otherwise the transfection rate will be low. For more product details, please visit Aladdin Scientific website.
① cell culture equipment, constant temperature incubator
② Phase contrast microscope
③ Centrifuge
Reagents:
① Donor plasmid pFastBac or pFastBacDUAL, DH5aDH10Bac, receptor cells Sf21 cells
② LB liquid, solid medium, Luria solid medium, SOC medium, Grace's culture medium
③ Restriction endonuclease
④ Ligase
⑤ Plasmid extraction kit
⑥ lipofectin