G-Quadruplex is a special DNA secondary structure and present in many important regulatory regions in human genome, such as the telomeric end and the promoters of some oncogenes. Specially, different forms of G-quadruplexes exist in telomeric DNA and c-myc promoter and play important roles in the pathway of cell proliferation and senescence. The effects of G-quadruplex ligands for either telomeric or c-myc G-quadruplex in vitro have been widely studied, but the specificity of these effects in vivo is still unknown. In the present research, various experiments were carried out to study the effect of G-quadruplex ligand SYUIQ-05 on tumor cell lines and the mechanism of this effect. Our results showed that it preferred to bind with G-quadruplex in c-myc and had rather insignificant effect on G-quadruplex in telomere. Therefore, it is possible that this compound had its antitumor activity for cancer cells mainly through its interaction with c-myc quadruplex.
Nanoluciferase
(Nluc), the smallest luciferase known, was used
as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection
of the aflatoxin B1 in cereal. Nanobody (clone G8) against
aflatoxin B1 was fused with nanoluciferase and cloned into
a pET22b expression vector, and then transformed into Escherichia
coli. The nanobody fusion gene contained a hexahistidine
tag for purification by immobilized metal affinity chromatography,
yielding a biologically active fusion protein. The fusion protein
G8-Nluc retained binding properties of the original nanobody. Concentration
of the coelenterazine substrate and buffer composition were also optimized
to provide high intensity and long half-life of the luminescent signal.
The G8-Nluc was used as a detection antibody to establish a competitive
bioluminescent ELISA for the detection of aflatoxin B1 in
cereals successfully. Compared to classical ELISA, this novel assay
showed more than 20-fold improvement in detection sensitivity, with
an IC50 value of 0.41 ng/mL and linear range from 0.10
to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can
be completed in one step within 2 h, from sample preparation to data
analysis. These results suggest that nanobody fragments fused with
nanoluciferase might serve as useful and highly sensitive dual functional
reagents for the development of rapid and highly sensitive immunoanalytical
methods.
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