Expression, purification and characterization of anti-α2δ1/NanoLuc fusion protein / 生物工程学报

The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG® M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.

Effects of honey to acyclovir in the rabbit eye transport kinetics / 中国中药杂志

<p><b>OBJECTIVE</b>Using pharmacokinetics to explore the mechanism of honey to enhance the efficacy of acyclovir (ACV) treatment of herpes simplex keratitis (HSK), providing the basis for combination of the prescription of two drugs and dosage regimen designed.</p><p><b>METHOD</b>Single dosages of 5% honey and 0% honey Meyasu eye ointment are injected into rabbit eyes. The aqueous humor of rabbit eye is measured at different times, specifically the content of ACV in aqueous humor by HPLC. Mathematical models are established, from which pharmacokinetic parameters are extracted and compared by mathematics and statistics methods.</p><p><b>RESULT</b>Both the 5% and 0% honey Meyasu eye ointment in rabbit eyes are belong to a two-compartment model. The absorption half-life of the 5% Meyasu eye ointment in aqueous humor is as 2.30 times longer, the distribution half-life is 2.12 times longer, the peak concentration is 1.17 times longer, the peak time is 1.36 times longer, AUC is 1.41 times longer when compared to the 0% Meyasu eye ointment.</p><p><b>CONCLUSION</b>Honey can significantly increase the ACV concentration and bioavailability in the eye, extend the action time of ACV in target cells and increase the retention capacity of ACV in the target tissue; thereby improving treatment success.</p>