To develop a magnetic nanoparticle chemiluminescence immunoassay (CLIA) for the determination of type Ⅰ procollagen N-terminal peptide (PINP) in human serum, we expressed a recombinant PINP-α1 protein in Corynebacterium glutamicum and used it as an immunogen to immunize BALB/c mice. We obtained three hybridoma cell lines that stably secret antibody against PINP-α1 protein. After further pairing and screening, we chose a monoclonal antibody 8C12 coupled with biotin as the capture antibody, and a monoclonal antibody 1F11 labeled horseradish peroxidase as the detection antibody. The antibodies combined with the serum samples, forming a sandwich complex which was used to detect the concentration of PINP in serum. After optimizing the conditions, we determined that the best working concentration of the capture antibody and the detection antibody were 3 μg/mL, and the incubation time was 30 minutes. The quantitative assay had a detection range of 5-1 100 ng/mL, with recovery rates between 93%-107% and the minimum detection limit of 1.22 ng/mL achieved. The intra-and inter-assay precisions were lower than 10%. The correlation coefficient of PINP results between this CLIA method and the Roche electrochemiluminescence immunoassay system was 0.906 2. Therefore, this CLIA method is specific and can be used to quantitatively detect the content of PINP in serum, which has the potential to become an auxiliary approach for bone disease examination.
Animals ; Humans ; Immunoassay ; Luminescence ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/isolation & purification* ; Procollagen/isolation & purification*

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naive Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.
Anti-HIV Agents/pharmacology ; Drug Resistance, Viral/*genetics ; HIV Envelope Protein gp41/*genetics/metabolism/pharmacology ; HIV Infections/virology ; HIV-1/*genetics/isolation & purification/*metabolism ; Humans ; Peptide Fragments/pharmacology ; *Polymorphism, Genetic ; Protein Structure, Tertiary/genetics ; Republic of Korea ; Virus Internalization

To identify human monoclonal HIV-l-neutralizing antibodies from an HIV-1 CRF07BC specific phage display antibody library by cell-based screening. 293T cells were transfected by pCH064. 2-Env plas mid and then used to biopan the phage antibody library. The positive phage clones were screened by cell based ELISA and sequenced for the variable region of heavy (VH) and light (VL) chains. The expressed Fabs were purified by Ni(+2) -NTA column and analyzed by SDS-PAGE. The cell- and gp120 protein-based ELISA as well as flow cytometry were used to measure Fab's binding activity. The neutralizing activity of Fabs was assessed by HIV-1 pseudoviruses. After 4-round biopanning, the binding phages to transfected cells were enriched about 650-folds. A total of 28 positive clones were screened out by cell ELISA and sequence analysis identified 5 different Fabs possessing unique VH and VL (2801, 2837, 2863, 2870 and 2920). Interestingly, these Fabs reacted with the Env-transfected 293T cells but not soluble gp120 proteins, suggesting that they might target conformation-dependent epitopes presenting on viral Env complex. We found that three Fabs (2801, 2863, 2870) exhibited potent neutralizing activity against CRF07_BC isolate CH120. 6 with IC50 of 2.24, 0.89 and 3.09 microg/mL respectively, and that 2801 and 2863 cross-neutral ized the subtype B isolate SF162 at IC50 of 0.69 and 3.52 microg/mL respectively. In conclusion, the HIV-1 Env-transfected 293T cells can be used to efficiently enrich and screen the phage antibody library and isolate human monoclonal HIV-1-neutralizing Fabs that target the Env complex-dependent conformational epitopes. Therefore, our studies provide a powerful platform for exploring the mechanism of HIV-1 neu tralizing response and for designing AIDS vaccines.
Antibodies, Monoclonal ; genetics ; immunology ; Antibodies, Neutralizing ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; HEK293 Cells ; HIV Antibodies ; genetics ; immunology ; HIV Envelope Protein gp120 ; genetics ; immunology ; HIV Infections ; immunology ; virology ; HIV-1 ; genetics ; immunology ; isolation & purification ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Neutralization Tests ; Peptide Library ; Transfection

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.
Animals ; Complementarity Determining Regions ; genetics ; immunology ; Cytokines ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Mice ; Peptide Library ; Protein Binding ; Receptors, Immunologic ; immunology ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification

Edible bird's nest contains lots of glycoproteins. The glycosylation inhomogeneity for glycoprotein often results in wide range of molecular weight and the difficulty for protein separation and charaterization. In this paper, proteins in the edible bird's nest were extracted using multiple extractions, and then digested by PNgase F and trypsin. The digest mixture was separated with HPLC, and peptides were identified based on MS/MS data searching. The results indicated that the extracted proteins were amount to 79.7% of total protein in the edible bird's nest. More than 20 species of peptides in the digested mixture were identified. The sequences of these peptides showed similarity with some proteins from Swiss-prot. The research indicated that deglycosylation, tryptic digestion coupled with HPLC-MS/MS is a proper strategy for characterization of proteins in the edible bird's nest.
Amino Acid Sequence ; Animals ; Birds ; Chromatography, High Pressure Liquid ; Glycoproteins ; chemistry ; Mass Spectrometry ; Medicine, Chinese Traditional ; Peptide Fragments ; chemistry ; isolation & purification ; metabolism ; Proteolysis

Tyrosinase is a key enzyme related to skin pigmentation disorders of elderly people, while self-aggregation of the amyloid-beta peptide, Abeta42, has been considered as a key event in the pathogenesis of Alzheimer's disease (AD). The present study was undertaken to investigate the inhibitory effects of 20 samples from Rhodiola species on tyrosinase and Abeta42 aggregation, and to isolate their corresponding bioactive components. The results demonstrated that the oligomeric proanthocyanidins (OPCs) commonly found in Rhodiola species were the major bioactive components corresponding to their anti-tyrosinase and anti-Abeta42 aggregation bioactivities. Salidroside, a representative compound of Rhodiola plants, proved not to be active in the present studies.
Amyloid beta-Peptides ; antagonists & inhibitors ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Glucosides ; isolation & purification ; pharmacology ; Monophenol Monooxygenase ; antagonists & inhibitors ; metabolism ; Peptide Fragments ; antagonists & inhibitors ; metabolism ; Phenols ; isolation & purification ; pharmacology ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Proanthocyanidins ; isolation & purification ; pharmacology ; Rhodiola ; chemistry

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.
Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Coronavirus ; chemistry ; immunology ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; isolation & purification ; Peptide Fragments ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests

This study is to explore whether the Wnt/beta-catenin signaling pathway is involved in the process of tripchlorolide (T4) protecting against oligomeric Abeta(1-42)-induced neuronal apoptosis. Primary cultured cortical neurons were used for the experiments on day 6 or 7. The oligomeric Abeta(1-42) (5 micromol x L(-1) for 24 h) was applied to induce neuronal apoptosis. Prior to treatment with Abeta(1-42) for 24 h, the cultured neurons were pre-incubated with T4 (2.5, 10, and 40 nmol x L(-1)), Wnt3a (Wnt signaling agonists) and Dkk1 (inhibitors) for indicated time. Then the cell viability, neuronal apoptosis, and protein levels of Wnt, glycogen synthase kinase 3beta (GSK3beta), beta-catenin and phospho-beta-catenin were measured by MTT assay, TUNEL staining and Western blotting, respectively. The result demonstrated that oligomeric Abeta(1-42) induced apoptotic neuronal cell death in a time- and dose-dependent manner. Pretreatment with T4 significantly increased the neuronal cell survival and attenuated neuronal apoptosis. Moreover, oligomeric Abeta(1-42)-induced phosphorylation of beta-catenin and GSK3beta was markedly inhibited by T4. Additionally, T4 stabilized cytoplasmic beta-catenin. These results indicate that tripchlorolide protects against the neurotoxicity of Abeta by regulating Wnt/beta-catenin signaling pathway. This may provide insight into the clinical application of tripchlorolide to Alzheimer's disease.
Amyloid beta-Peptides ; antagonists & inhibitors ; toxicity ; Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; Diterpenes ; isolation & purification ; pharmacology ; Female ; Fetus ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Neurons ; cytology ; drug effects ; Neuroprotective Agents ; isolation & purification ; pharmacology ; Peptide Fragments ; antagonists & inhibitors ; toxicity ; Phenanthrenes ; isolation & purification ; pharmacology ; Phosphorylation ; Plants, Medicinal ; chemistry ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tripterygium ; chemistry ; Wnt Proteins ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo express and purify the fusion proteins of glutathione S-transferase (GST)-N-terminal of histone deacetylase4 (HDAC4-N') (1-1952 bp) and GST- C-terminal of HDAC4 (HDAC4-C') (1708-3255 bp) in E.coli.

METHODSThe DNA fragments (HDAC4-N' and HDAC4-C') amplified by PCR were ligated into GST fusion vector (pGEX-6P-1) to construct the recombinant plasmids. After identification with restriction digestion and DNA sequencing, the recombinant plasmids were transformed into E.coli BL21 and induced by IPTG for their expression. After identification by SDS-PAGE and Western blotting, the target proteins were purified by glutathione sepharose 4B.

RESULTSThe results of restriction digestion and DNA sequencing confirmed successful construction of the recombinant plasmids. The relative molecular masses of the fusion proteins were approximately 110500 and 93080 as shown by SDS-PAGE. Western blotting demonstrated that the fusion proteins could be recognized by the specific anti-HDAC4 antibody.

CONCLUSIONWe have successfully constructed the recombinant expression vectors of pGEX-6P-1/HDAC4-N' and pGEX-6P-1/HDAC4-C' and induced the expression of the fusion proteins, which may facilitate functional studies of HDAC4 with other proteins.

Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Histone Deacetylases ; biosynthesis ; genetics ; Humans ; Peptide Fragments ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Repressor Proteins ; biosynthesis ; genetics

To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.
Amyloid beta-Peptides ; genetics ; immunology ; Antibody Specificity ; immunology ; Humans ; Peptide Fragments ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification