MicroRNA-141 regulates the expression level of ICAM-1 on endothelium to decrease myocardial ischemia-reperfusion injury.

A growing number of studies have suggested microRNAs (miRNAs) are involved in the modulation of myocardial ischemia-reperfusion (MI/R) injury; however, the role of endogenous miRNAs targeting endothelial cells (ECs) and its interaction with ICAM-1 in the setting of MI/R remain poorly understood. Our microarray results showed that miR-146a, miR-146b-5p, miR-155*, miR-155, miR-497, and miR-451 were significantly upregulated, whereas, miR-141 and miR-564 were significantly downregulated in the ECs challenged with TNF-α for 6 h. Real-time PCR analyses additionally validated that the expression levels of miR-146a, miR-155*, and miR-141 were consistent with the microarray results.

Construction and evaluation of DNA vaccine encoding Hantavirus glycoprotein N-terminal fused with lysosome-associated membrane protein.

Background: Hantaviral diseases can have a high case fatality rate within the absence of broadly effective antiviral treatments or vaccines. We developed a DNA vaccine targeting the Hantavirus glycoprotein N-terminal (Gn) to major histocompatibility complex class II compartment by fusing the antigen with lysosome-associated membrane protein 1 (LAMP1), which altered antigen presenting pathway and activated the CD4+ T cells.

Methods: The segments of Gn and LAMP1 were cloned into vector pVAX1, and recombinant plasmid was constructed by inserting Gn sequence into LAMP1, between luminal and the transmembrane/cytoplasmic domains.

Down regulation of TRAIL and FasL on NK cells by Cyclosporin A in renal transplantation patients.

TNF-related apoptosis-inducing ligand (TRAIL) and FasL can participate in cell mediated cytotoxicity via their death domain-mediated apoptotic signaling in the host-versus-graft disease occurred after renal transplantation. However, the effect of Cyclosporin A (CsA) commonly used as a drug to prevent and to treat renal transplant rejection, on these molecules have not been fully determined. In the present study, we found that with CsA administration, the expression of TRAIL and FasL predominantly on NK cells from renal transplantation patients was increased at day 5 after operation and went down to normal level on day 13.

[Prokaryotic expression, purification and identification of NY-ESO-1/GST fusion protein in E.coli].

Aim: To construct an expression plasmid for NY-ESO-1 gene and identify the expression of recombinant protein NY-ESO-1/GST in E.coli.

Methods: NY-ESO-1 segment was amplified from the testis cDNA library by RT-PCR and cloned into the prokaryotic expression vector pGEX4T-1 downstream tagged by GST to construct the expression plasmid pGEX-4T1-NY-ESO-1.

[The application of quantitative detection of cystatin C in evaluation of renal function after renal transplantation].

Aim: To establish ELISA method for quantitate the concentration of cystatin C (cys C) and to monitor the renal function of patients before and after renal transplantation.

Methods: Hybridomas secreting monoclonal antibodies (mAbs) against human cys C were produced and sandwich ELISA kit for quantitatively detecting cys C was established. Then the concentrations of serum cystatin C (Scys C) and urine cystatin C (Ucys C) from normal controls and 23 patients undergoing renal transplantation were detected and their relationship with serum creatinine (SCR) was analyzed.

Murine monoclonal antibodies generated against mouse/rat hemokinin-1.

The mouse/rat hemokinin-1 (m/rHK-1) was discovered nearly 9 years ago. This molecule is a peptide comprising 11 amino acids. The m/rHK-1 was found to be mainly expressed in central immune organs like bone marrow, and was proven to have lymphopoietic roles in B and T lymphocyte development.

Cancer/testis antigen CT45: analysis of mRNA and protein expression in human cancer.

CT45 is a cancer/testis gene that we previously identified by massively parallel signature sequencing. Encoded by a multigene family on chromosome X, CT45 showed restricted mRNA expression to normal testis and various cancers. In this study, monoclonal antibodies were generated against recombinant CT45 protein, and CT45 protein expression in normal and tumor tissues was evaluated by immunohistochemical analysis.

Application of sandwich ELISA for detecting tag fusion proteins in high throughput.

Based on a series of mAbs against four frequently used tags--the human Ig Fc fragment, GST, maltose-binding protein, and thioredoxin--we developed corresponding sandwich enzyme-linked immunosorbent assay (ELISA) to detect these tag fusion proteins. As a supplement for Western blot, the successfully established ELISA was specific, sensitive, quantitative, easy to perform, time-saving, and last but not least, suitable for high-throughput screening of tag fusion proteins. Determination of soluble tag fusion proteins expressed by various systems with the sandwich ELISA developed in the present study could be a valuable and promising tool for the wide application of tag-protein fusion systems in the rapidly growing field of proteomics research.

[Prokaryotic expression, purification and identification of VEGFR2 D3.4/GST fusion protein in E.coli].

Aim: To construct a prokaryotic expression vector for the expression of VEGFR2 D3.4/GST fusion protein, Express and purify the fusion protein.

Methods: The coding sequence of the third and fourth extracellular domain of human VEGFR2 gene fragment was synthesized and subcloned into pGEX4T-1 vector downstream of the GST fragment, an E.

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio).

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit.

Generation of rat monoclonal antibodies against murine LAIR-1.

The leukocyte-associated Ig-like receptor 1 (LAIR-1), an inhibitory receptor bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIM), is expressed on the majority of peripheral leukocytes, including NK cells, T cells, B cells, monocytes, dendritic cells, granulocytes, and thymocytes and is involved in immunologic regulation and hematopoiesis. Murine LAIR-1 (mLAIR-1) is the homolog molecule of human LAIR-1. Using mLAIR-1-Fc as the immunogen and the technique of rat B lymphocyte hybridoma, we raised three hybridoma cell lines secreting monoclonal antibodies (MAb) to mLAIR-1, designated FMU-mLAIR-1.

[Preparation and characterization of monoclonal antibodies against human CD112 (Nectin2/PRR2)].

Aim: To express and purify fusion protein of human CD112 extracellular region, prepare monoclonal antibodies (mAb) to CD112 and investigate the distribution of CD112 molecule.

Methods: The CD112-Fc fusion protein was expressed with gene recombinant technique in eukaryotic system and purified with affinity chromatography column. The BALB/c mice were immunized with purified CD112-Fc for preparation of mAb by hybridoma technique.

[Preparation of the monoclonal antibodies against human BORIS and the expression pattern of BORIS in normal and diseased human mammary tissues].

Aim: To prepare monoclonal antibodies(mAb) to BORIS antigen and analyze its expression pattern in breast cancer, benign breast disease and normal breast tissues.

Methods: Female BALB/c mice were immunized with recombination human BORIS protein. Hybridoma cell lines were established by hybridoma technique.

Characterisation of monoclonal antibodies to the TNF and TNF receptor families.

Tumour necrosis factor (TNF) family ligands and their corresponding receptors play important roles in the immune system and are involved in immune regulation such as lymphoid development, cell proliferation, differentiation, activation and death. Antibodies against these ligands and receptors together with Fc-fusion proteins, have been particularly useful as immunological tools in addressing the underlying involvement of these proteins in these contexts and furthermore, have given us hope in using them as potential therapeutic agents. Over last few years, there have been many additions to these ever-growing TNF family ligands and their receptors.

[Regulation of soluble TRAIL and membrane TRAIL in Jurkat cells by PMA].

Aim: To investigate the regulation of soluble and membrane bound TNF-related apoptosis-inducing ligand (TRAIL) in Jurkt cells by phorbol myristic acctate (PMA), and the cytotoxicity of the two forms of TRAIL.

Methods: Jurkat cells were cultured in the presence of 40 ng/mL PMA for different time. The production of sTRAIL was determined by ELISA, and expression of mTRAIL was analyzed by indirect fluorescence staining and flow cytometry analysis.

[Apoptosis-inducing ligand TRAIL can be recruited to lipid rafts].

Aim: To investigate the relationship between tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) and cell membrane microdomain lipid rafts.

Methods: The expression of TRAIL on K562 cells was detected by indirect immunofluorescence staining and flow cytometry. The lipid rafts on K562 cells were detected with FITC-labeled cholera toxin B subunit (CTx-FITC) which bound to the GM1 glycosphingolipid in lipid rafts.

[Construction and expression of human EpCAM eukaryotic expression vectors and identification of their products].

Aim: To construct EpCAM eukaryotic expression vectors pIg-EpCAM and pEGFP-EpCAM and to express them in COS7 cells.

Methods: EpCAM cDNA was amplified by PCR and then inserted into the pIg and pEGFP vectors respectively to construct recombinant vectors pIg-EpCAM and pEGFP-EpCAM. The two recombinant vectors were transfected into COS7 cells under the mediation of liposome.

[The inhibition of TNF-induced NF-kappaB nuclear translocation in ECV304 cells by mAbs against human TNF].

Aim: To identify the inhibition of TNF-induced NF-kappaB nuclear translocation by three anti-human TNF mAbs, D2, E6 and F6.

Methods: TNF solutions were pretreated with mAbs D2, E6 and F6 as well as control mAb at 37 degrees Celsius for 1 h, respectively, and then they were added to ECV304 cell cultures. After 1 hour, the cells were harvested and nuclear proteins were extracted.

[Preparation and characterization of monoclonal antibodies against rat Nogo molecule].

Aim: To prepare monoclonal antibodies against rat Nogo molecule and study the distribution of Nogo in rat central nervous system (CNS).

Methods: Murine mAbs were prepared by hybridoma technique. The distribution of Nogo in rat CNS was identified by immunofluorescent histochemical staining.

Preparation and characterization of a set of monoclonal antibodies to TRAIL and TRAIL receptors DR4, DR5, DcR1, and DcR2.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo 2L) is a novel cytotoxic ligand belonging to TNF superfamily. Among TRAIL receptors, death receptor 4 (DR4) and DR5 containing death domain (DD) in their cytoplasmic region mediate apoptosis-signaling upon TRAIL binding, while decoy receptor 1 (DcR1) and DcR2 with a truncated or non-functional DD play "decoy" role. The interaction of TRAIL and TRAIL receptors plays important roles both in immunoregulation and immune pathogenesis of some diseases.