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| − | <li><a href="#ove">Overview</a></li> | + | <li><a href="#epro">Envelope Protein</a></li> |
| − | <li><a href="#int">Introduction</a></li> | + | <li><a href="#cle">CLEC5A</a></li> |
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| − | <a href="#mec">Mechanism</a>
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| − | <li><a href="#nov">Liquid Sample without Virus</a></li>
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| − | <li><a href="#vi">Liquid Sample with Virus</a></li>
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| − | </ul>
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| − | </li>
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| | <div class="contents s12 m6 l4" id="text"> | | <div class="contents s12 m6 l4" id="text"> |
| | <article> | | <article> |
| − | <section id="ove"> | + | <section id="epro"> |
| − | <h2>Overview</h2> | + | <h2>Envelope Protein (E protein)</h2> |
| − | <p>Dengue fever has been a severe problem in Taiwan for a long time, but there is still no vaccine to prevent it and no medicine to cure it. Therefore, we tried to tackle this problem by producing the peptides of the tandem repeat sequences (PTRSs) that can bind to the envelope protein (E protein) of the dengue virus, providing an alternative opportunity for detection and prevention.</p> | + | <p>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.</p> |
| | <br> | | <br> |
| − | <p>Currently, no dengue virus detection kits are made in Taiwan. Thus, our team decided to develop a detection kit for dengue virus. We used PTRSs to capture the targets in our detection kit instead of commonly used antibodies. The following are the advantages of PTRSs compared to antibodies.</p> | + | <div id="imginfo"> |
| − | <ul> | + | <img width="80%" src=""> |
| − | <li><b>Lower cost</b></li>
| + | <p> Figure 6. E protein with two HA tags, matching the expected size of 1,551 bp. M = DNA marker.</p> |
| − | <p> PTRSs are shorter and have a simpler structure than antibodies. Thus, the production costs of PTRSs are much lower.</p> | + | </div> |
| − | <li><b>Longer storage</b></li>
| + | <br> |
| − | <p>Because PTRSs are linear and short sequence, they are not easily denatured. </p>
| + | <p> 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. |
| − | <li><b>Fast production</b></li> | + | 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.</p> |
| − | <p>The production of antibodies requires injection of antigens into a mouse or a rabbit. After injection, several months are required to produce the antibodies. This procedure is too complex and time consuming. We use linear array epitope (LAE) to produce PTRSs, which can greatly reduce the production time, especially with the short sequences and simple structures of PTRSs.</p> | + | <br> |
| − | <li><b>Easy modification</b></li>
| + | <div id="imginfo"> |
| − | <p>A big advantage of PTRSs is that they can be modified easily. If the dengue virus mutates, we can rapidly change PTRSs to detect the mutated strain.</p> | + | <img width="80%" src=""> |
| − | </ul> | + | <p>Figure 7. Plasmid extraction of pET-29a(+), matching the expected size of about 5,000 bp. M = DNA marker.</p> |
| | + | </div> |
| | + | <div id="imginfo"> |
| | + | <img width="80%" src=""> |
| | + | <p>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.</p> |
| | + | </div> |
| | </section> | | </section> |
| | <br> | | <br> |
| − | <section id="int"> | + | <section id="cle"> |
| − | <h2>Introduction</h2> | + | <h2>CLEC5A</h2> |
| − | <p>We use a lateral flow immunochromatographic assay like the one used in a pregnancy test, mounted with PTRSs to detect the dengue virus. Our detection kit consists of the sample pad, conjugate pad, test line, control line, and absorbent pad. The sample pad and absorbent pad are made of cellulose fibers, the conjugate pad is made of glass fiber and the test line and the control line are on the other glass fiber. The functions of these pads are:</p> | + | <p>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. |
| − | <ul>
| + | 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 DH5which 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.</p> |
| − | <li>Sample pad is where the liquid sample is placed.</li>
| + | |
| − | <li>Conjugate pad is where the dengue virus reacts with PTRSs.</li>
| + | |
| − | <li>Test line indicates a positive result.</li>
| + | |
| − | <li>Control line ensures that the detection kit is reliable.</li>
| + | |
| − | <li>Absorbent pad absorbs water and prevents the backflow.</li>
| + | |
| − | </ul>
| + | |
| − | </section>
| + | |
| − | <br>
| + | |
| − | <section>
| + | |
| − | <div id="mec">
| + | |
| − | <h2>Mechanism</h2>
| + | |
| − | <p>Two PTRSs that can bind with the dengue virus E protein are required. The first PTRSs (PTRS-1) is attached to gold nanoparticles and placed on the conjugate pad. The second PTRSs (PTRS-2) is placed on the test line. E proteins from the dengue virus are expressed and conjugated on the control line.</p>
| + | |
| − | <br>
| + | |
| − | <p>Since both PTRSs bind to the E proteins, if present, dengue virus will bind to PTRS-1 which are attached to the gold nanoparticles on the conjugate pad, then bind to PTRS-2 on the test line. PTRS-1, with the attached nanoparticles, will always bind to the E proteins on the control line.</p>
| + | |
| − | <br>
| + | |
| − | <p>Gold nanoparticles appear red when they aggregate, and the red color is visible with the naked eye, as shown in Figure 1.</p>
| + | |
| − | <div id="imginfo">
| + | |
| − | <img width="80%" src="https://static.igem.org/mediawiki/2020/4/49/T--CCU_Taiwan--Design_0.png">
| + | |
| − | <p>Figure 1. Components of the detection kit</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | <br> | | <br> |
| − | <div id="nov"> | + | <div id="imginfo"> |
| − | <h3>Liquid Sample without Virus</h3> | + | <img width="80%" src=""> |
| − | <p>Figure 2 shows how the detection kit works when the liquid sample does not contain dengue virus. Because there are no virus particles in the liquid sample, PTRS-1 conjugated on gold nanoparticles bind to nothing and flow past the test line. When flowing to the control line, where the E proteins are located, PTRS-1 bind to the E proteins. An aggregation of gold nanoparticles attached to PTRS-1 leads to a red band on the control line.</p> | + | <p>Figure 9. CLEC5A with a Myc tag, matching the expected size of about 600 bp. M = DNA marker.</p> |
| | </div> | | </div> |
| | <div id="imginfo"> | | <div id="imginfo"> |
| − | <img width="80%" src="https://static.igem.org/mediawiki/2020/6/69/T--CCU_Taiwan--Design1_1.gif"> | + | <img width="80%" src=""> |
| − | <p>Figure 2. Mechanism in absence of dengue virus</p> | + | <p>Figure 10. Plasmid extraction of pET-29b(+), matching the expected size of about 5,000 bp. M = DNA marker.</p> |
| | + | </div> |
| | + | <div id="imginfo"> |
| | + | <img width="80%" src=""> |
| | + | <p>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.</p> |
| | </div> | | </div> |
| | <br> | | <br> |
| − | <div id="vi"> | + | <p>pET-29b(+)_CLEC5A was then transformed to BL21(DE3) and induced to express protein with 1.8 ml bacteria and 1 M 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.</p> |
| − | <h3>Liquid Sample with Virus</h3> | + | <br> |
| − | <p>Figure 3 shows how the detection kit works when the liquid sample contains dengue virus. PTRS-1 conjugated on gold nanoparticles bind to the virus particles in the conjugated pad. When the virus particles reach the test line, the unbound virus surface can interact with PTRS-2 attached to the test line. These restricted virus particles lead to an aggregation of gold nanoparticles, which results in a red band indicating a positive result. The residual gold nanoparticles then flow to the control line. A red band can be found on the control line also due to the aggregation of gold nanoparticles.</p> | + | <div id="imginfo"> |
| | + | <img width="80%" src=""> |
| | + | <p>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.</p> |
| | </div> | | </div> |
| | <div id="imginfo"> | | <div id="imginfo"> |
| − | <img width="80%" src="https://static.igem.org/mediawiki/2020/b/b7/T--CCU_Taiwan--Design2_1.gif"> | + | <img width="80%" src=""> |
| − | <p>Figure 3. Mechanism in presence of dengue virus</p> | + | <p>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.</p> |
| | + | </div> |
| | + | <br> |
| | + | <p>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.</p> |
| | + | <br> |
| | + | <div id="imginfo"> |
| | + | <img width="80%" src=""> |
| | + | <p>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.</p> |
| | </div> | | </div> |
| − | </section>
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| − | <br>
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| − | <hr>
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| − | <section id="ref" style="word-break:break-all; word-wrap:break-all;">
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| − | <h4>References</h4>
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| − | <p>Baeumner, Antje J.; Schlesinger, Nicole A.; Slutzki, Naomi S.; Romano, Joseph; Lee, Eun Mi; Montagna, Richard A. Biosensor for dengue virus detection: Sensitive, rapid, and serotype specific. American Chemical Society. Doi: 10.1021/ac015675e<br>
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| − | Horstick, Olaf; Tozan, Yesim; Wilder-Smith, Annelies. Reviewing Dengue: Still a Neglected Tropical Disease? PLOS Neglected Tropical Diseases. Doi: 10.1371/journal.pntd.0003632<br>
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| − | Lim, Jong Min; Kim, Ji Hong; Ryu, Myung Yi; Cho, Chae Hwan; Park, Tae Jung; Park, Jong Pil. An electrochemical peptide sensor for detection of dengue fever biomarker NS1. Analytica Chimica Acta. Doi: 10.1016/j.aca.2018.04.005<br>
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| − | Mazbahul Kabir; Syamal Raychaudhuri; James William Needham; Stanislaw Morkowski. Lateral Flow Assaysystemand Methods Forts Use. United States Patent and Trademark Office. Patent No.: US 8, 399, 261 B2<br>
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| − | Wang, Hsi Kai; Tsai, Cheng Han; Chen, Kuan Hung; Tang, Chung Tao; Leou, Jiun Shyang; Li, Pi Chun; Tang, Yin Liang; Hsieh, Hsyue Jen; Wu, Han Chung; Cheng, Chao Min. Cellulose-based diagnostic devices for diagnosing serotype-2 dengue fever in human serum. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Doi: 10.1002/adhm.201300150
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| − | </p>
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| | </section> | | </section> |
| | </article> | | </article> |
