Team:GreatBay SCIE/Engineering
Production and Specificity Verification of scFv with Genetic Engineering Tools
scFv amino acid sequence is obtained from Dr. Dong Wei’s work which selected anti-ɑ-amanitin scFv by biopanning and phage display technology[1]. We cloned this sequence into plasmid vector pET28b under the T7 promoter system and added a 6*His tag that allows IMAC affinity purification to be done. After finding out the scFv has formed inclusion bodies instead of the soluble expression, we reconstructed the expression system, with the addition of a pelB tag. This particular tag can provide scFv secretion to the periplasm, making the extraction process easier and more convenient.
We successfully verified the production of scFv by anti-His tag colloidal gold test strips and SDS-PAGE.
Fig.1.Construction of pelB scFv expression vector.
The anti-His tag colloidal gold test strips apply a competitive test, which means the positive result is given by one red control line, and the negative result is given by two red lines, both the control line and the test line.
Fig.2. Colloidal gold test strip verification.
The SDS-PAGE showed scFv at the target size - 28kDa, for our cell pellets.
Fig.3.The SDS-PAGE showing the expression of scFv in BL21(DE3)-pET28b-pelB-scFv.
The binding ability and sensitivity of our scFv were also tested, by ELISA - Enzyme-Linked ImmunoSorbent Assay. The scFv from periplasm is first extracted by the arginine extraction method and then used for ELISA and indirect competitive ELISA(ic-ELISA). OD450 was measured for the final color change, and scFv showed exceptional binding ability and sensitivity by the calculation of IC50. IC50 is a measure of the toxin concentration for 50% inhibition, and the lower the IC50, the higher the binding sensitivity. Our IC50 is 13.27ng/ml, which is a very low value.
Fig.4.The result showing scFv ELISA when toxin is present and not present.
Fig.5. IC50 value calculation.
All in all, the improvement, construction of scFv, and the verification of production and binding was successful.
The Selection and Binding Specificity Test of Aptamers
The manufacturing of aptamers is simpler compared to antibodies, as the production of aptamers does not involve the use of bacteria and any organisms, also safer to use.
Selection of Aptamers (SELEX)
SELEX (Systematic Evolution of Ligands by EXponential enrichment) is used to select aptamers of the correct 3-dimensional shape in a group of random aptamers, of the same basepair length. Aptamers with low affinity are washed off, conserving those with relatively high specificity, and use PCR to amplify this pool, used for the next round of experiments. 15~20 cycles of selection are repeated, purifying the aptamers with the highest affinity to our toxins.
We chose an approach of SELEX that combines the magnetic beads. Toxin-magnetic beads conjugates are made, and aptamers are added to the solution. Those with higher specificity bind with the toxins immobilized on the magnetic beads and others remain free in the solution. A magnetic rack is used to separate magnetic beads and elutes. Elutes which contain low affinity-aptamers are discarded. Aptamers on the immobilized toxins are washed off and sent for PCR amplification, making a pool for the next round of selection. The pool is gradually purified and in the end, it will contain aptamers with extremely high specificity. After each round, we use a small amount of the pool of each round to do gel electrophoresis. This is known as a control PCR, in which we are able to see if the extraction of aptamers is successful.
We finished the 15 cycles of SELEX against beta-amanitin, the 15th control PCR is shown. Its band locates at 80 bp, and it is a very bright strand, which means we succeeded in conserving those aptamers.
Verification of Aptamer Specificity (ELONA)
To verify the specificity of the aptamer pools selected by SELEX[1]. ELONA (Enzyme-Linked OligoNucleotide Assay) is carried out using 96 deep well plate to test the binding specificity of aptamers to immobilized toxins. ELONA is very similar to ELISA and it also measures the value of OD450 in positive correlation where the higher the OD450 is, aptamers can be concluded to have higher binding specificity to toxins.
To further expand and enrich our project, we repeated the ELONA of the aptamers of amanitin, the aptamers are directly synthesized using the aptamer sequence from an article published in 2016 [2], which they have already completed the selection and binding specificity test of their aptamers. Our ELONA test was very successful as the alpha-BSA conjugate presented a very high OD450, indicating that our ELONA approach fits our aptamers and targets. This also proves the reliability of the data of ELONA of the aptamers of beta-amanitin.
References
- Muszyńska K, Ostrowska D, Bartnicki F, Kowalska E, Bodaszewska-Lubaś M, Hermanowicz P, Faulstich H, Strzałka W. Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides. Acta Biochim Pol. 2017;64(3):401-406. doi: 10.18388/abp.2017_1615. Epub 2017 Aug 9. PMID: 28787470.
- Zhang X, He K, Zhao R, Feng T, Wei D. Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors. Food Anal Methods. 2016. doi:10.1007/s12161-016-0509-3
- Han Q, Xia X, Jing L, et al. Selection and characterization of DNA aptamer specially targeting α-amanitin in wild mushrooms. SDRP J Food Sci Technol. 2018;3(6):497-508. doi:10.25177/JFST.3.6.2