Difference between revisions of "Team:JNFLS/Design"
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<h1>Design</h1> | <h1>Design</h1> | ||
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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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| − | < | + | <b>Reference:</b> |
| − | < | + | <p>[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.</p> |
| − | < | + | <p>[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.</p> |
| − | < | + | <p>[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.</p> |
| − | < | + | <p>[4]Zhou hui. Studies on competitive mechanism triggered signal amplification based aptasensors [D]. Hunan University,2009.</p> |
| − | </ | + | <p>[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.</p> |
| + | <p>[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.</p> | ||
| + | <p>[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.</p> | ||
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Latest revision as of 22:53, 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.
[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.