Linear Array Epitope
The results of template-repeated PCR (TR-PCR), shown in Figure 1., indicate that the length of the tandem-repeated sequence (TRS) varies with the concentration of the primer. We found the typical trend that the lower the concentration of primer, the higher the number of repeats of the TR-PCR products. However, the product concentration decreases when the primer concentration increases. In our project, the optimal primer concentrations are 0.2 μM for TRS-151*7 and 0.08 μM for TRS-110*3.
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Figure 1. The TR-PCR results of (a) TRS-151*7 and (b) TRS-110*3. The values listed on each represent the concentration of primer (in μM). The yellow arrow shows the typical ladder pattern of TR-PCR. M = DNA marker.
TRS-110*3 and TRS-151*7 obtained from TR-PCR were transferred to adaptor PCR (AD-PCR). The ladders shown in Figure 2. are the products obtained from AD-PCR. The sequences ranging in size from 200-400 bp and 50-200 bp, identified as TA-151 and TA-110, respectively, were extracted and ligated to TA vectors. Mutiple bands found in the results of AD-PCR due to the fact that there were several sites available for ligation of the adapter. Therefore, various lengths of sequences were generated during AD-PCR. These ligated sequences were then successfully transformed into DH5α competent cells. The colonies shown in Figure 3. suggest the bacteria survives due to the antimicrobial peptides (AMP) in the TA vector.
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Figure 2. The results of AD-PCR of (a) TRS-151*7 and (b) TRS-110*3. The value listed on each lane represents the concentration of primer (in μM). M = DNA marker.
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Figure 3. The culture plates suggest that TA-151 (a) and and TA-110 (b) were successfully transformed into DH5α competent cells.
The results of colony PCR show that the sizes of TRS-151*7 (Figure 4. (a)) and TRS-110*3 (Figure 4. (b)) are estimated to be 420 bp and 300 bp, respectively.
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Figure 4. Colony PCR of (a) TRS151*7 and (b) TRS110*3. The sizes of TRS-151*7 and TRS-110*3 are estimated to be 420 bp and 300 bp (including the M13 primers and some sequences on the TA-vector), respectively. The numbers above the lanes indicate colony number. M = DNA marker.
Plasmids from these colonies were extracted, and TRS were obtained using the restriction sites of Ncol and Hindlll on the TA vector. TRS-151*7 and TRS-110*3 have sizes of 200-300 bp (Figure 5.a) and 90-100 bp (Figure 5.b), respectively, which fits our expectations.
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Figure 5. The result of digestion of (a) TA-151 and (b) TA-110, which have sizes of 200-300 bp and 90-100 bp, respectively. M = DNA marker.
Finally, these TRSs were extracted and ligated to pET-29b(+). However, we ran into a problem during transformation of pET-29b(+) into DH5α. Currently, we are still seeking to optimize the transformation conditions.
Envelope Protein (E protein)
A plasmid containing the whole structural protein of the dengue virus was obtained from National Health Research Institute. The Envelope protein (E protein) with two HA tags from the plasmid were amplified using PCR. Figure 6. shows that the sequence of the E protein with the HA tags were amplified, matching the expected size of 1,551 bp.
Figure 6. E protein with two HA tags, matching the expected size of 1,551 bp. M = DNA marker.
We prepared pET-29a(+) as the vector for expressing E protein and confirmed in Figure 7. that plasmid extraction shows the size of pET-29a(+) is about 5,000 bp, which is close to the theoretical size, 5,371 bp. We attempted to transform the pET-29a(+)_E protein into DH5α and check the transformation using colony PCR. However, Figure 8. shows the results not what was expected, and the size of the plasmid was wrong, so the ligation and transformation failed. We are trying to fix this.
Figure 7. Plasmid extraction of pET-29a(+), matching the expected size of about 5,000 bp. M = DNA marker.
Figure 8. Colony PCR of pET-29a(+)_E protein, which only has a size of about 300 bp rather than the expected 500 bp. M = DNA marker. P = positive control, pET-29b(+)_CLEC5A.
CLEC5A
A plasmid containing the CLEC5A extracellular domain with a Myc tag was obtained from OriGene Technologies. The CLEC5A extracellular domain and Myc tag were amplified using PCR. Figure 9. shows that the sequence of the CLEC5A extracellular domain with a Myc tag were amplified successfully, with a size of 564 bp. pET-29b(+) was prepared successfully as a vector, as shown in Figure 10. , with a theoretical size of 5,370 bp. pET-29b(+)_CLEC5A was obtained by inserting the CLEC5A into pET-29b(+) and then transforming it into DH5α which was confirmed using colony PCR. Figure 11. shows the size of CLEC5A extracellular domain with Myc tag, and T7 promoter and terminator is about 900 bp, close to the theoretical size of 904 bp.
Figure 9. CLEC5A with a Myc tag, matching the expected size of about 600 bp. M = DNA marker.
Figure 10. Plasmid extraction of pET-29b(+), matching the expected size of about 5,000 bp. M = DNA marker.
Figure 11. Colony PCR of pET-29b(+)_CLEC5A, which has an expected size of about 900 bp including the CLEC5A extracellular domain and Myc tag and T7 promoter and terminator. M = DNA marker.
pET-29b(+)_CLEC5A was then transformed to BL21(DE3) and induced to express protein with 1.8 ml bacteria and 1 mM IPTG for 2 hours. Figure 12. shows the results from four different colonies with and without induction. The strong bands from 28 to 35 kDa indicate expression of the CLEC5A extracellular domain with the Myc tag, which has a size of 33 kDa. The results were also confirmed using Western blot based on the HA tag, which is part of pET29b(+). Figure 13. shows that the anti-His tag binds to the His tag, suggesting that the protein expression was successful.
Figure 12. SDS-PAGE of CLEC5A from a small-scale culture. After induction with IPTG, the strong bands at about 33 kDa indicate the expressing of CLEC5A. M = protein marker. NI = Non-Induction. I = Induction.
Figure 13. Western blot of CLEC5A. An anti-His tag was used to bind to the His tag on the CLEC5A protein. The stronger band on the induction lanes suggests the experiments were successful. NI = Non-Induction. I = Induction.
Finally, we expressed CLEC5A on a large scale. The bacterial culture was lysed using a French press, then separated with a high-speed centrifuge. Figure 14. indicates CLEC5A is always found in the pellets no matter how long they were induced.
Figure 14. SDS-PAGE of CLEC5A large-scale expression. The protein was induced for 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours, separately. The bacterial solution was lysed with a French press and separated with a high-speed centrifuge. The results indicate CLEC5A is always expressed in the pellet under these conditions. M = protein marker. Sup. = supernatant.
