Difference between revisions of "Team:JNFLS/Design"

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In the very beginning of our project, we brainstormed about the detection measures for HCV. The current detection method used in all blood centers in Shandong province is ELISA-based HCV antibody detection, which caused the awkward situation that not even a single positive result was gained in the last decade. HCV antibody detection will be the past, but what the future will be? ELISA-based antigen detection? No, the detection must be sufficiently sensitive, what if the amount of virus in the blood is too low? Nucleic acid detection? No, it shares the same drawback as the previous method; also, RNA is way easier to be degraded. In the end, the works of Zhang et al.[3] and Zhou[4] inspired us. We eventually designed a biosensor based on aptamer and rolling circle amplification.  
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HCV C protein gene is constructed into the pColdII vector and expressed in E.Coli. Then ssDNA aptamer library is constructed. HCV C protein is used to screen the nucleic acid aptamer by SELEX technology. Using the competing reaction of the target antigen, the adapter sequence, padlock probe and complementary sequence of aptamer, a highly sensitive fluorescent adapter sensor is developed based on the rolling circle replication. When there is no target antigen, the complementary sequence binds with aptamer probe instead of the padlock probe, which triggers rolling circle amplification reaction. Whereas when the aptamer binds with the target antigen, the complementary sequence hybridizes with the padlock probe. Under the action of DNA ligase, the padlock probe is further cyclized and a rolling circle amplification occurs under the action of DNA polymerase. So, it is facilitated to detect HCV in blood. By designing different aptamer sequences and related nucleic acid sequences, the sensing system can be used as a general method to detect another targets antigen.
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<img src="https://static.igem.org/mediawiki/2018/b/b3/T--JNFLS--ban.jpg"style="width:70%">
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<img src="https://static.igem.org/mediawiki/2018/b/b6/T--JNFLS--htak.png"style="width:30%">
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<p>Mechanism of our device.</p>
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<p>The aptamer is originally bound with its partially complementary sequence; thus, it remains double-stranded before the detection. If target antigen, or say, HCV Core protein, exists, since the affinity between the aptamer[5] and HCV Core protein is stronger than that between the aptamer and its complementary sequence, the aptamer will bind to the antigen and leave the complementary sequence in the reaction system. The complementary sequence will bind to the padlock probe which contains the complementary sequence on both sides of its gap, and “sew” the gap. Then, after the treatment of DNA ligase, the complementary-sequence-padlock-probe complex will be encircled. Next, under the function of DNA polymerase Phi29[6], the rolling circle amplification[7] can initiate. After adding the quenching fluorescence group, the signal is able to be detected. </p>
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<img src="https://static.igem.org/mediawiki/2018/d/d7/T--JNFLS--tian.jpg"style="width:70%">
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<p>Primer for HCVCO120 gene.</p>
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<img src="https://static.igem.org/mediawiki/2018/7/73/T--JNFLS--xian.jpg"styel="width:70%">
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<p>Primer for HCVCO173 gene.</p>
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<img src="https://static.igem.org/mediawiki/2018/a/a0/T--JNFLS--baobao.jpg"styel="width:1%">
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<p>pCold II plasmid.</p>
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<p>After the experiment began, we were in an urge to find an efficient way to gain HCV core protein. HCV core protein originally consists of 191 amino acid, which is hard to be expressed in prokaryotic cells. However, Li [1] and Wu [2] found in their studies that HCV core protein can be expressed more rapidly and operably if the protein is truncated into 119, 125, or 173 amino acid in size. The HCVC protein we used consists of 120AA and 173AA, based on the works mentioned above. The result showed that the amount of protein expressed increase.
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<img src="https://static.igem.org/mediawiki/2018/7/7e/T--JNFLS--liu.jpg"style="width:80%">
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<img src="https://static.igem.org/mediawiki/2018/2/25/T--JNFLS--han.jpg"style="width:30%">
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Revision as of 16:12, 17 October 2018

Design

HCV C protein gene is constructed into the pColdII vector and expressed in E.Coli. Then ssDNA aptamer library is constructed. HCV C protein is used to screen the nucleic acid aptamer by SELEX technology. Using the competing reaction of the target antigen, the adapter sequence, padlock probe and complementary sequence of aptamer, a highly sensitive fluorescent adapter sensor is developed based on the rolling circle replication. When there is no target antigen, the complementary sequence binds with aptamer probe instead of the padlock probe, which triggers rolling circle amplification reaction. Whereas when the aptamer binds with the target antigen, the complementary sequence hybridizes with the padlock probe. Under the action of DNA ligase, the padlock probe is further cyclized and a rolling circle amplification occurs under the action of DNA polymerase. So, it is facilitated to detect HCV in blood. By designing different aptamer sequences and related nucleic acid sequences, the sensing system can be used as a general method to detect another targets antigen.

Reference:

[1]Li Shengtao. The expression and purification of the truncated HCV core protein (HCV Core125) and its antibody preparation [D]. Kunming University of technology,2012.

[2]Wu Xianbo. Cloning, expression of a gene fragment encoding HCV core antigen and purification, antigenicity analysis of the recombinant protein [D]. First military medical university of the people's liberation army,2003.

[3]Zhang Songbai, Zheng Liying, Hu Xia, Shen Guangyu, Liu Xuewen, Shen Guoli, Yu Ruqin. Highly sensitive fluorescent aptasensor for thrombin detection based on competition triggered rolling circle amplification [J]. Chinese Journal of Analytical Chemistry,2015,43(11):1688-1694.

[4]Zhou hui. Studies on competitive mechanism triggered signal amplification based aptasensors [D]. Hunan University,2009.

[5]Shi S, Yu X, Gao Y, et al. Inhibition of hepatitis C virus production by aptamers against the core protein[J]. Journal of Virology, 2014, 88(4): 1990-1999.

[6]Dean F B, Nelson J R, Giesler T L, et al. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification[J]. Genome research, 2001, 11(6): 1095-1099.

[7]Banér J, Nilsson M, Mendel-Hartvig M, et al. Signal amplification of padlock probes by rolling circle replication[J]. Nucleic acids research, 1998, 26(22): 5073-5078.