Search

Objective: To explore the thermal damage to epithelial cell adhesion molecule(EpCAM)-positive tumor cells by novel aptamer-guided magnetic nanoparticles(AptNPs). Methods: EpCAM aptamer SYL3C was connected to NPs via biotin-streptavidin reaction. The diameter of AptNPs were characterized by Dynamic Light Scattering(DLS). The binding feature of the aptamer to EpCAM-positive tumor cells was evaluated by Prussian blue dyeing. Thermal damage under alternative magnetic field was measured bylactate dehydrogenase (LDH). The apoptosis of EpCAM-positive tumor cells was detected by acridine orange/ethidium bromide (AO/EB) double staining. Results: The average size of AptNPs was 282 nm. Flow cytometry and Prussian blue dyeing showed that AptNPs exhibited strong binding to the EpCAM-positive tumor cells but not to the EpCAM-negative tumor cells. Moreover, when incubated with 1.5×10(8) AptNPs under alternative electromagnetic fieldfor 5 hours, the viability of EpCAM-positive HCT116 cells and A549 cells was 28.9% and 54.4%, respectively, significantly lower than 76.7% of EpCAM-negative HepG2 cells ( P <0.05). Conclusions: AptNPs can improve the thermal damage to EpCAM-positive tumor cells, and may have potential utility in the development of tumor targeted therapy.

Photoaffinity labeling is widely applied to demonstrate targets of small molecule ligands. In this paper, biotin photoaffinity labeled molecule with propargyl group 1 has been designed and synthesized, followed it's labeling of N2-acetyl-2'-O-propargyl guanosine 9 by "click chemistry". This technology presents delight development potential in labeling of second messenger cyclic nucleotide, antisense oligonucleotide or siRNA.

To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.

Targeting drug delivery system (TDDS) is one of the most concerned research fields in cancer treatment because it can bind selectively and react with the target diseased sites at the cellular or sub-cellular level, making distribution and release of drugs in a controlled manner, thus enhance therapeutic effects and reduce toxic and side-effects on normal cells. Polyamidoamine dendrimer (PAMAMD) is a kind of newly developed polymer in nanometer degree. Hyper-branched, monodispersity, three-dimensional structure and host-guest entrapment ability make it used as drug carrier, gene delivery system and imaging agent. Various targeting ligands, which have high affinity to specific organs, tissues or cells in human body, can be linked to surface functional groups of PAMAMD. And drugs and theoretical gene are carried by encapsulation or chemical conjugation. Finally, PAMAMD targeting drug delivery system can carry drugs and theoretical gene to diseased sites and then release them for targeted therapy. The PAMAMD-based conjugates have small size, ligh permeability and retention effect (EPR), low toxicity and so on. The research progress of PAMAMD modified by different ligands in targeting drug delivery system is reviewed, and research direction of the PAMAMD targeting delivery system in the future is also suggested.

Objective: To cross-link the McAb 1A2E1 to BNHS reagents and apply this biotinylated McAb to detect AIB1 antigen of target cells., Methods: The McAb 1A2E1 was mixed with BNHS reagents to generate Biotin-1A2E1. The competitive inhibition ELISA and immunocytochemical method were applied to detect the biologic activity of antibody., Results: The antibody kept high biological activities (with a titer of 1 : 3200) and sensitivities in detecting the AIB1 protein of breast cancer cell., Conclusion: The method of preparing biotinylated McAb is successful, and the prepared biotinylated McAb can be applied to detect target cells which express AIB1 antigen. This McAb provide useful tool for tumor auxiliary diagnosis.

In this experiment, a novel biotin-avidin conjugation probe was synthesized and employed in the detection of reverse-phase protein microarray. Firstly, the proportion of the biotin-avidin conjugation probe was optimized. Then the rat IgG and goat anti-rat IgG system was served as a model to optimize the fabrication conditions of reverse-phase protein microarray, including the non-specific absorption of streptavidin-Cy3 molecules, spotting buffer as well as protein activities. At last, the biotin-avidin conjugation probe was applied to the detection of the reverse-phase protein microarray. The results show that the protein microarray prepared by using BSA spotting buffer could prevent non-specific absorptions of fluorescent molecules and improve the sensitivity, effectively. In addition, compared with traditional biotin-avidin system, the detection limit could be improved four times using the biotin-avidin conjugation probe. In conclusion, the biotin-avidin conjugation probe has its merits of easy synthesis, low price and could be further conjugated with other signal amplification techniques, which is promising to be used in the detection of protein microarray.

Objective: To establish a new method for the study of DNA-protein interaction., Methods: Six SPF BALB/c mice were divided into two groups, the lipopolysaccharide (LPS)-treated group (n=3) and the normal control group (n =3). The LPS-treated mice were subjected to tail vein-injection of LPS (25 mg/kg) to reproduce an endotoxic shock model. The biotin-labeled DNA probe for the LPS inducible gene (LIG) promoter was amplified by polymerase chain reaction (PCR). The cell nuclear extracts from liver of mice were prepared and mixed together. DNA pull-down assays were performed by using magnetic beads conjugated with streptavidin. Proteins binding on the beads were eluted by salts with different concentrations. The samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and displayed by silver-stain to compare differential bands between two groups., Results: Fifteen proteins in the nuclear extracts were found to be different between the mice of two groups. Analysis of the differential bands of SDS-PAGE displayed that, compared with normal control group, there were four proteins increased and one protein declined in the mice treated with LPS when eluted with low salt. While under the condition of high affinity elution, there were nine proteins increased and one disappeared after LPS treatment., Conclusion: In this study, a novel method was set up by combining the biotin-streptavidin system and magnetic beads isolation technique, providing a new way to study DNA-protein interaction. This strategy is helpful for further understanding the regulation of gene expression with a view of "omics".

Aim: To establish a novel method for antibody analysis based on visual protein microarray., Methods: The antibodies spotted on the aldehyde-modified slides were incubated with corresponding antigens labeled by biotin, followed by silver enhancement detection method, and the chromogenic images were scanned by CCD camera., Results: The affinity and specificity of each antibody was assayed by this method and six antibodies were found to have higher specificity than the others. A detection limit of 0.4 microg/L was obtained for anti-globulinIIIC013, anti-albumin HPSIIB007, and anti-albumin HPSIIIB007., Conclusion: This method is simple, fast and visual. In addition, it needs less sample amount than traditional immunoassay and has potential to achieve high throughput.

Biotinylated chitosan nanoparticles (Bio-CS-NP) were prepared for the active delivery to cancer cells and its characterization was investigated in this study. The preparation process included two steps. First, biotinylated chitosan ( Bio-CS ) was obtained through a reaction between sulfosuccinimidobiotin and chitosan (CS). Second, Bio-CS-NP were prepared by the precipitation of Bio-CS with sodium chloride solution. With a biotin reagent box, the conjugation densities of biotin on the surface of Bio-CS-NP were determined. The morphology and diameter of the nanoparticles were assayed by transmission electron microscope (TEM) and laser light scattering particle analyzer, respectively. The uptake of nanoparticles by human hepotacarcinoma HepG2 cells, for example, Bio-CS-NP and chitosan nanoparticles (CS-NP) without any modification, was quantitatively examined. The results indicated that the conjugation densities of biotin on the surface of Bio-CS-NP were 2.2 biotin CS. Bio-CS-NP were spherical, smooth on the surface. The average diameter was 296.8 nm. The polydispersion index was 0.155. The uptake of Bio-CS-NP by HepG2 cells was much higher than that of CS-NP (P < 0.05). It demonstrated that Bio-CS-NP can be applied as a new vehicle to actively deliver anticancer drugs to tumor cells. The method for the determination of biotin was simple and practical.

Objective: To compare two kinds of labeled streptavidin biotin(LsAB) methods and provide the optimal method of antigen retrieval., Methods: The alkaline phosphatase(AP) LsAB method and the horseradish peroxidase (HRP) LsAB method were used to stain anti-tryptase in paraffin embedding tissue of patients with chronic obstructive pulmonary disease (COPD) and their staining effects were compared. The antigens were repaired by high pressure cooking, tryptin digestion and microwave, and the repairing effects were compared., Results: The backgrounds of stained sections were more distinct and the color distinction of nucleus and positive signal were brighter by AP-LsAB, compared with those by HRP-LsAB; the repairing effect of high pressure cooking was better than the repairing effect of tryptin digestion and than that of microwave., Conclusion: AP-LsAB and high pressure cooking retrieval were recommended for use in immunohistochemical staining of pulmonary inflammatory tissue.

Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases.