The secondary structures of fusion protein pp150/MDBP, including alpha-helix, beta-sheet, turn regions, were analyzed by Garnier-Robson's and Chou-Fasman's methods; the antigenic epitopes of B cells were analysed by using hydrophilicity plot. The results showed that the fusion protein pp150/MDBP might have less alpha-helix, but be rich in beta-sheet and turn regions. The epitopes recognized by B cells may be at 7-56 amino acid residues or adjacent to 137-192 amino acid residues.
Amino Acid Sequence ; Binding Sites ; Cytomegalovirus ; chemistry ; Epitopes ; Humans ; Molecular Sequence Data ; Phosphoproteins ; chemistry ; immunology ; Protein Structure, Secondary ; Viral Fusion Proteins ; chemistry ; immunology ; Viral Matrix Proteins ; chemistry ; immunology

OBJECTIVE: To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide (LPS). METHODS: Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope. RESULTS: SPLUNC1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells. CONCLUSION SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.
Cell Line, Tumor ; Glycoproteins ; isolation & purification ; pharmacology ; Humans ; Membrane Proteins ; chemistry ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Phosphoproteins ; isolation & purification ; pharmacology ; Pseudomonas aeruginosa ; drug effects ; Respiratory Mucosa ; chemistry ; immunology ; Respiratory System ; chemistry ; immunology ; Transfection

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.
Animals ; Antibodies, Anti-Idiotypic/immunology/metabolism ; *Aptamers, Nucleotide/chemistry/immunology ; Biological Assay ; *Cotinine/administration & dosage/chemistry ; Flow Cytometry ; Hep G2 Cells ; Humans ; Mice ; NIH 3T3 Cells ; Phosphoproteins/*chemistry/immunology ; Protein Binding ; RNA-Binding Proteins/*chemistry/immunology ; *Vascular Endothelial Growth Factor A/chemistry/immunology

Human cytomegalovirus (HCMV) infection is an ubiquitous herpesvirus disease in human populations. It is rarely pathogenic to healthy adults, yet it may cause severe outcome to organ transplant recipients, the immunocompromised individuals and pregnant women. Using DNA from HCMV AD169 strain as template, the UL32 gene encoding pp150 protein fragment and the UL57 gene encoding MDBP protein fragment were amplified by PCR technique. After the construction of cloning vector pMD18-T-UL32, pMD18-T-UL57, pMD18-T-UL32/UL57 and expression vector pET-11a-UL32/UL57, the recombinant fusion proteins pp150/MDBP were induced with IPTG in BL21 host strain. The results showed that the relative molecular weight of recombinant fusion proteins pp150/MDBP is about 27 kD, the product of fusion proteins takes 17.45% in the total proteins in host bacteria, the analytical result was positive to the fusion proteins pp150/MDBP via Western blot technique, while the purified recombinant fusion proteins have strong antigenicity detected by ELISA and protein chip compared with whole virus antigens from HCMV. It was demonstrated that when used for the detection of serum from the clinical patients it has the same detection rate compared with the whole virus antigen. It needs further research for application.
Cytomegalovirus ; DNA-Binding Proteins ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Peptide Fragments ; blood ; genetics ; immunology ; isolation & purification ; Phosphoproteins ; chemistry ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; blood ; genetics ; immunology ; isolation & purification ; Transcription Factors ; chemistry ; Viral Matrix Proteins ; chemistry

PRAS40 (proline-rich Akt substrate 40 kD) associates with mammalian target of rapamycin complex 1(mTORC1), serine 183 site (Ser183) of PRAS40 can be phosphorylated by mTORC1. To prepare the phosphorylated PRAS40 (Ser183) antibody, We chosen 10-amino acid including Ser183 as antigen peptide through antigenicity and hydrophobicity analysis, hinged on keyhole limpet hemocyanin (KLH), and used the KLH-peptide to immunize rabbits. After antibody serum titer detection by enzyme linked immunosorbent assay (ELISA), the antibody was purified with rProtein A sepharose fast flow and dephosphorylated antigen membrane. The antibody titrate reached 1:10 000 after purification and its special property was enhanced with absorption treatment of dephosphorylated antigen membrane. In addition, we used rabbit anti-PRAS40 antibody and the phosphorylated PRAS40 (Ser183) antibody to detect PRAS40 expression in several cell lines, including the normal cells HL7702, HEK293, tumor cells HepG2, A549 and S180. There were no quite difference among these cells; otherwise, we observed the decreased phosphorylation level of Ser183 after amino acid withdrawal treatment. Therefore, the polyclonal phosphorylated PRAS40 (Ser183) antibody was specific to PRAS40 (Ser183) site and could be used for the function study of PRAS40.
Adaptor Proteins, Signal Transducing ; Animals ; Antibodies ; analysis ; Cell Line, Tumor ; Humans ; Male ; Mechanistic Target of Rapamycin Complex 1 ; Multiprotein Complexes ; Phosphoproteins ; chemistry ; immunology ; Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rabbits ; Serine ; metabolism ; TOR Serine-Threonine Kinases