Team:JNFLS/Design
Design
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.
Mechanism of our device.
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.
Primer for HCVCO120 gene.
Primer for HCVCO173 gene.
pCold II plasmid.
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.
[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.