Team:NJTech China/Experiments
EXPERIMENTS
1. Production of mouse IL-2-RFP fusion protein
1) Construction of plasmid pGAP-ss-mIL2-RFP
Plasmid pGAP-ss-mIL2-RFP was synthesized by GENEWIZ. The sequence of mIL2-RFP was optimized for Pichia pastoris and a 6xHis tag for protein purification was attached to the C-terminus of the sequence.
We added the DH5α E.coli containing plasmid pGAP-mIL2-RFP synthesized by GENEWIZ into LB liquid meidum containing 1% kanamycin, and incubated them at 37℃, 2000rpm overnight.
We extracted the plasmid from E.coli with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by microvolume UV-Vis spectrophotometer. We recorded the concentration for further experiments.
2) Transformation of Pichia
We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid from circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a electrophoresis to ensure whether the digestion had been completed. If only one strip of the target size appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction Kit. The concentration of linearized plasmid was measured for further experiments.
We prepared yeast competent cells according to the procedures in our Protocol. Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock. After kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L G418 and incubated these plates at 30℃.
3) Screening positive transformants
After 3 days’ culture, some colonies appeared on the screening plates. We chose some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of mIL2 and KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of transformation. If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours incubation at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction Kit and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed genome as a template and ss-mIL2 F and ss-mIL2 R as primers, genomic PCR was performed to verify whether our target gene was successfully integrated into the yeast genome. After successful validation, we submitted the genomic samples to GENEWIZ for sequencing to further confirm the sequence.
4) Fermentation
If the sequencing results match the designed sequence, we will take some recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of incubation, we transformed some cultures into fermentation medium and incubated them in oscillating incubator at 200 rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the fermentation solution at 4℃, waiting for product collection and purification.
5) Product collection and purification
We dispersed the fermentation solution in order to get supernatant. We gave the supernatant a ultrafiltration. Since our produced protein is about 44kDa, we chose a 30kDa filter to ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did the affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further purified product. After each step of single processing, we used SDS-PAGE to detect the effect of extraction and purification of the extracted and residual samples.
2. Production of mouse IL-2 protein
1) Construction of plasmid pGAP-mIL2-RFP
We used the primers ss-mIL2 F and ss-mIL2 R to copy the fragment of mIL2 with alpha signal sequence from plasmid pGAP-ss-mIL2-RFP by PCR. Since we had added these primers homogenous arm of XbaⅠregion in plasmid pGAP-KanR, so we could put our fragment into plasmid by one step cloning later. We used OMEGA Gel Extraction Kit to obtain fragments from PCR solution. We used FastDigest Xba Ⅰ to linearize the plasmid and extract linearized plasmid with OMEGA Gel Extraction Kit as well. Then we connected ss-mIL2 to plasmid using Vazyme ClonExpress II fast Cloning Kit. We used this cloning solution to transform competent DH5α E.coli cells according to the procedures in our Protocol. Afterwards we screened the positive cells using LB medium plate containning 0.1% Kana. We transformed some of colonies into 0.1% Kana LB liqiud medium. After incubated at 37℃ for hours, these cultures were used for extraction of plasmid by AXYGEN AxyPrep Plasmid Miniprep Kit and recombinant E.coli cells were stored in 30% glycerin YPD solution at -20℃. We used primers to copy ss-mIL2 from extracted plasmid. We ran a electrophoresis to ensure that ss-mIL2 exsits in plasmid and sent PCR solution to GENEWIZ for sequencing.
According to the sequencing report supported by GENEWIZ, we knewn that we had gotten E.coli containing plasmid with correct ss-mIL2 sequence. We added it into 1% LB liquid medium and extracted plasmid from cultures later. The concentration of plasmid was measured by microvolume UV-Vis spectrophotometer. We recorded the concentration for further experiments.
2) Transformation of Pichia
We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid from circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a electrophoresis to ensure whether the digestion had been completed. If only one strip of the target size appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction Kit. The concentration of linearized plasmid was measured for further experiments.
We prepared yeast competent cells according to the procedures in our Protocol. Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock. After kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L G418 and incubated these plates at 30℃.
3) Screening positive transformants
After 3 days’ culture, some colonies appeared on the screening plates. We chose some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of mIL2 and KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of transformation. If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours incubation at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction Kit and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed genome as a template and ss-mIL2 F and ss-mIL2 R as primers, genomic PCR was performed to verify whether our target gene was successfully integrated into the yeast genome. After successful validation, we submitted the genomic samples to GENEWIZ for sequencing to further confirm the sequence.
4) Fermentation
If the sequencing results match the designed sequence, we will take some recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of incubation, we transformed some cultures into fermentation medium and incubated them in oscillating incubator at 200 rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the fermentation solution at 4℃, waiting for product collection and purification.
5) Product collection and purification
We dispersed the fermentation solution in order to get supernatant. We gave the supernatant a ultrafiltration. Since our produced protein is about 19kDa, we chose a 10kDa filter to ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did the affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further purified product. After each step of single processing, we used SDS-PAGE to detect the effect of extraction and purification of the extracted and residual samples.
3. Production of human IL-2-RFP fusion protein
1) Construction of plasmid pGAP-ss-hIL2-RFP
Plasmid pGAP-ss-hIL2-RFP was synthesized by GENEWIZ. The sequence of hIL2-RFP was optimized for Pichia pastoris and a 6xHis tag for protein purification was attached to the C-terminus of the sequence.
We added the DH5α E.coli containing plasmid pGAP-hIL2-RFP synthesized by GENEWIZ into LB liquid meidum containing 1% kanamycin, and incubated them at 37℃, 2000rpm overnight.
We extracted the plasmid from E.coli with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by microvolume UV-Vis spectrophotometer. We recorded the concentration for further experiments.
2) Transformation of Pichia
We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid from circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a electrophoresis to ensure whether the digestion had been completed. If only one strip of the target size appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction Kit. The concentration of linearized plasmid was measured for further experiments.
We prepared yeast competent cells according to the procedures in our Protocol. Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock. After kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L G418 and incubated these plates at 30℃.
3) Screening positive transformants
After 3 days’ culture, some colonies appeared on the screening plates. We chose some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of hIL2 and KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of transformation. If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours incubation at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction Kit and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed genome as a template and ss-hIL2 F and ss-hIL2 R as primers, genomic PCR was performed to verify whether our target gene was successfully integrated into the yeast genome. After successful validation, we submitted the genomic samples to GENEWIZ for sequencing to further confirm the sequence.
4) Fermentation
If the sequencing results match the designed sequence, we will take some recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of incubation, we transformed some cultures into fermentation medium and incubated them in oscillating incubator at 200 rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the fermentation solution at 4℃, waiting for product collection and purification.
5) Product collection and purification
We dispersed the fermentation solution in order to get supernatant. We gave the supernatant a ultrafiltration. Since our produced protein is about 40kDa, we chose a 30kDa filter to ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did the affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further purified product. After each step of single processing, we used SDS-PAGE to detect the effect of extraction and purification of the extracted and residual samples.
4. Production of TAT(rigid linker) fusion protein
1) Construction of plasmid pGAP-TAT(rigid linker)
Plasmid pGAP-ss- TAT(rigid linker) was synthesized by GENEWIZ. The sequence of TAT(rigid linker) was optimized for Pichia pastoris and a 6xHis tag for protein purification was attached to the C-terminus of the sequence.
We added the DH5α E.coli containing plasmid pGAP- TAT(rigid linker) synthesized by GENEWIZ into LB liquid meidum containing 1% kanamycin, and incubated them at 37℃, 2000rpm overnight.
We extracted the plasmid from E.coli with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by microvolume UV-Vis spectrophotometer. We recorded the concentration for further experiments.
2) Transformation of Pichia
We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid from circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a electrophoresis to ensure whether the digestion had been completed. If only one strip of the target size appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction Kit. The concentration of linearized plasmid was measured for further experiments.
We prepared yeast competent cells according to the procedures in our Protocol. Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock. After kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L G418 and incubated these plates at 30℃.
3) Screening positive transformants
After 3 days’ culture, some colonies appeared on the screening plates. We chose some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of TAT and KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of transformation. If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours incubation at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction Kit and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed genome as a template and ss-TAT F and ss-TAT R as primers, genomic PCR was performed to verify whether our target gene was successfully integrated into the yeast genome. After successful validation, we submitted the genomic samples to GENEWIZ for sequencing to further confirm the sequence.
4) Fermentation
If the sequencing results match the designed sequence, we will take some recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of incubation, we transformed some cultures into fermentation medium and incubated them in oscillating incubator at 200 rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the fermentation solution at 4℃, waiting for product collection and purification.
5) Product collection and purification
We dispersed the fermentation solution in order to get supernatant. We gave the supernatant a ultrafiltration. Since our produced protein is about 55kDa, we chose a 30kDa filter to ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did the affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further purified product. After each step of single processing, we used SDS-PAGE to detect the effect of extraction and purification of the extracted and residual samples.
5. Production of TAT(soft linker) fusion protein
1) Construction of plasmid pGAP-TAT(soft linker)
Plasmid pGAP-ss- TAT(soft linker) was synthesized by GENEWIZ. The sequence of TAT(soft linker) was optimized for Pichia pastoris and a 6xHis tag for protein purification was attached to the C-terminus of the sequence.
We added the DH5α E.coli containing plasmid pGAP- TAT(soft linker) synthesized by GENEWIZ into LB liquid meidum containing 1% kanamycin, and incubated them at 37℃, 2000rpm overnight.
We extracted the plasmid from E.coli with AXYGEN AxyPrep Plasmid Miniprep Kit. Then the concentration of plasmid was measured by microvolume UV-Vis spectrophotometer. We recorded the concentration for further experiments.
2) Transformation of Pichia
We prepared a digested system containing FastDigest BamH Ⅰ to cut plasmid from circinate into linear. This system was incubated at 37℃ for about an hour. Then we made a electrophoresis to ensure whether the digestion had been completed. If only one strip of the target size appears on the agarose gel, we could recycle all of the linearized plasmid with OMEGA Gel Extraction Kit. The concentration of linearized plasmid was measured for further experiments.
We prepared yeast competent cells according to the procedures in our Protocol. Then we mixed linearized plasmid and competent Pichia in a system and gave it a electric shock. After kept at 30℃ for 2 h, we painted the system on YPD solid medium containing 0.3g/L, 0.6g/L and 1g/L G418 and incubated these plates at 30℃.
3) Screening positive transformants
After 3 days’ culture, some colonies appeared on the screening plates. We chose some of them to do colony PCR. We used two kinds of primer designed to copy the sequences of TAT and KanR. After reaction, we gave the PCR solution a electrophoresis to judge the result of transformation. If it appears to a right brand, we transform this colony into 1% Kana YPD medium. After hours incubation at 30℃, we extracted yeast genome from culturing fluid using Solarbio Yeast Genomic DNA Extraction Kit and preserved these recombinant yeasts in 30% glycerin YPD solution at -20℃. Using the proposed genome as a template and ss-TAT F and ss-TAT R as primers, genomic PCR was performed to verify whether our target gene was successfully integrated into the yeast genome. After successful validation, we submitted the genomic samples to GENEWIZ for sequencing to further confirm the sequence.
4) Fermentation
If the sequencing results match the designed sequence, we will take some recombinant yeasts from the cryotube and add them to the YPD liquid medium. After hours of incubation, we transformed some cultures into fermentation medium and incubated them in oscillating incubator at 200 rpm, 30℃. We recorded the OD600 every 24 hours until the value became stable. Finally we stored the fermentation solution at 4℃, waiting for product collection and purification.
5) Product collection and purification
We dispersed the fermentation solution in order to get supernatant. We gave the supernatant a ultrafiltration. Since our produced protein is about 55kDa, we chose a 30kDa filter to ultrafilter our samples. As a result, we can get our protein in the solution above membrane. We did the affinity chromatography with packed nickel column on the ultrafiltered sample to obtain a further purified product. After each step of single processing, we used SDS-PAGE to detect the effect of extraction and purification of the extracted and residual samples.
Validation
1. Proliferation effect on lymphocyte
(1) CCK8 proliferation assay
The proliferation effect of IL-2 on CD3+ T cell is evaluated by CCK8 assay, where 5×104 cells/100 µl/well is seeded in 96-well plate. Next, 100µl of complete media (RPMI1640 which is supplemented with FBS 10% and ConA 2.5 µg/ml) containing various concentrations of IL-2 (0.3, 1, 3, 10, 30, 100 and 300 nM), as a positive control, is added and incubated for 72h in 37 °C. Afterwards, 20µl CCK8 is added to each well and incubate for 4h in 37 °C. Optical density is measured at 450 nm on a Microplate reader.
(2) CFSE staining proliferation assay
CFSE staining proliferation assay is performed using carboxyfluorescein succinimidyl ester (CFSE) (BD bioscience, USA) staining. CD3+ T cell are stained by 10 µM CFSE and incubated for 20 min at 37℃. After washing by RPMI 1640 containing 10%FBS (Gibco, Brazil), cells are seeded into the 24-well plate at 1.0×106 cells/ml in the fresh media containing ConA 2.5 µg/ml, with different concentrations of IL-2 (0，0.3, 1, 3, 10, 30, 100 and 300 nM) for incubation at 37℃. After 10 days, the cells are analyzed by FACS Calibur Flowcytometry (BD Biosciences, USA), and data are analyzed by FlowJo 7.6.2 software (FlowJo, LLC, CA). To determine cell proliferation, lymphocytes are gated according to their forward and side scatters (SSC). To compare the rounds of proliferation in various culture conditions, cell populations with different levels of CFSE intensity (dilutions) are considered as a generation.
2. specifically binding of NJT0401 and PD-L1 on tumor cells
MDA - MB - 231 cells are seeded into 3 24-well plates at 2.0×104 cells/ml. Then cells are treated with 10ng/ml INF-γ for 24h. 500µl of complete media (RPMI1640 which is supplemented with FBS 10%) containing various concentrations of fusion protein (0.01nM, 0.1Nm, 1nM, 10nM, 100Nm, 1000nM), is separately added and incubated in 37℃. For the second plate, 500µl of complete media (RPMI1640 which is supplemented with FBS 10% ) containing various concentrations of anti-PD-L1(0.01nM, 0.1Nm, 1nM, 10nM, 100Nm, 1000nM), as a positive control, is separately added and incubated in 37℃. For the third plate, 500µl of complete media(RPMI1640 which is supplemented with FBS 10% ) , as a blank control, is added and incubated in 37℃.After 24h incubation avoiding light, the cells are washed with PBS twice and test with FACS Calibur Flowcytometer (BD Biosciences, USA). (rabbit anti-human pd-l1 monoclonal antibody (E1L3N) bound with phycoglobin (PE) is used to prepare samples. As a control, the cell samples are stained for 30 minutes with rabbit IgG monoclonal antibody (DA1E) conjugated with PE. The cell count of each sample is >105. The data is analysed by FlowJo 7.6.2 software (FlowJo, LLC, CA)
3. Sustained drug releasing model：
BSA is used as a model protein drug to study the drug release behavior. Analyze the hydrogels drug release kinetics of bovine serum protein (BSA) with different crosslinking degrees in vitro, and explore whether hydrogels loaded with drugs can achieve the effect of slow release.
Proliferation of T cells is determined in vitro using a BrdU proliferation ELISA kit (Roche). To investigate cell proliferation in vivo , TAT are injected into the left rear foot pad of tumor bearing mice .3 d later,tumor-bearing mice received an injection of 50 mg BrdU/kg of body weight. Animals are killed 18 h after the last injection. CD8+ T cells are isolated from the thymus, spleen by MACS as described above. The percentages of BrdU+ CD8+T cells are determined by flow cytometry.
An in situ polymerization method is used to incorporate BSA molecules into hydrogel.Briefly, BSA is initially dissolved in PBS to get a 0.02 wt% solution, and then HACHO and γ-PGA-ADH are dissolved in 0.02 wt% BSA solution, respectively. After that, these two solutions are mixed to prepare the BSA-loaded hydrogels at room temperature. The drug release tests are performed by immersing BSA-loaded hydrogels in 80 mL PBS, then placed in a circular mold and sealed with a sealing membrane ,which occasionally shaking at 37℃. At every 4 hours, the release medium (0.2 mL) is taken out and replaced with an equal volume of PBS, the tests lasted 72 hours. The BCA protein concentration assay kit is used to determine the total amount of BSA by color reaction.
All release experiments are performed in triplicate.
4. Anti-tumor effect and long-term immune memory effect of fusion protein (a perspective).
Mice are inoculated subcutaneously with 5*105 4T1 cells and randomly divided into four groups. After inoculation, they are individually treated with (1) PBS, (2) γGlu (3) γGlu and fusion protein, (4) fusion protein and then the primary tumors are surgically resected on day 28. Four weeks later, these mice are re-challenged with 4T1 cells in the right flank. The distant tumor size and survival are measured every 2 days, and the experimental end point is defined as either death or a tumor size greater than 1000 mm3. The primary and distant tumors are imaged by IVIS Spectrum (PerkinElmer, USA) after intraperitoneal injection with d-Luciferin sodium salt at a dose of 3 mg/mouse.
To investigate cell proliferation in vivo, NJT0401 are injected into the left rear foot pad of tumor bearing mice. Three days later, tumor-bearing mice received an injection of 50 mg BrdU/kg of body weight. Animals are performed euthanasia 18 hours after the last injection. CD8+ T cells are isolated from the thymus, spleen by MACS as described above. The percentages of BrdU+ CD8+T cells are determined by flow cytometry.
2.5*105 4T1 tumor cells are orthotopically implanted into Balb/c mice. When tumor volumes reach to 50-100mm3, mice are randomly divided into 4 groups, (1)PBS, (2)γGlu, (3)γGlu and fusion protein, (4)γGlu+anti-PDL1 (n=8). Treat i.p.at days 10, 12, 14 and 16, respectively. Twenty-four hours after the last treatment, carry out intratumoral analysis of surface PD-L1 accumulation.