Identification of epitope recognized by mAb 15 A11 sepecific against cartilage oli-gomeric matrix protein / 中国免疫学杂志

Objective:To identify the epitope of mAb15A11 which is specific against RA associated autoantigen cartilage oligomeric matrix protein ( COMP ).Methods: A filamentous phage library displaying random linear dodecapeptides was used to mapping the epitope of mAb15A11.After three rounds of screenings,40 phage clones were selected at random and sequenced.The specificity of phages was confirmed by enzyme immunoassays.Homology search by ClustalW2 and structure analysis by PyMol to identified the epitope amino acid sequence.Western blot analysis of COMP and ELISA analysis of COMP-derived peptides were used to confirm epitope′s characterization.Results: After repeated screenings using bio-panning method, 2 clones were identified, which interacted specifically with mAb 15A11.Homology search did not find succession consensus sequence within COMP molecular,which indicated that the epitope was not linear.PyMol Structure analysis identified the rationality of conformational epitope.Western blot analysis and ELISA of EDTA-treated COMP further prove an conformational structure of the epitope recognized by mAb 15A11.ELISA analysis of COMP-derived peptides demonstrated both disulfide bonds between 229 C-243 C and 237 C-253 C and every epitope amino acid of 232 G,238 H,240 H,241 A,244 V,247 R and 251 R were essential to the binding of mAb 15A11 with COMP.Conclusion: In this study, the potential B cell antigentic epitopes of mAb 15A11 was identified by phage display library.The epitope amino acids sequence and char-acterization were also recognized.It may have important theoretical value for the study of reaction mechanism of COMP antibody and antigen and may also show application significance in the detection of rheumatoid arthritis.

Analysis of gene structure, cloning and expression of cyp51 from Ustilago maydis / 生物工程学报

The cyp51 primers and two pairs of mutant primers which removed different transmembrane region were designed based on Ustilago maydis cyp51 gene structure analysis. The full cyp51 DNA fragment as well as mutant cyp51 genes were amplified and cloned by using the total DNA from Ustilago maydis as template, then subcloned into different expression vectors. The recombinant expression plasmids were transformed into Escherichia coli BL21 (DE3), BL21 (DE3) pLysS and Rosetta (DE3) respectively. A series of experiments leads to the finding that only pET32-YH-35 could be highly expressed at the optimal condition of 30 degrees C induced with 0.5 mmol/L IPTG The expressed protein (CYP51) showed biological activity by spectra analysis of the protein binding to 4 standard fungicides and to 14 XF-synthetic fungicide compounds, and only one XF-synthetic fungicide compound (XF-113) was similar to standard fungicides in binding constant. This compound is promising to be a new effective antifungal drug. These results will facilitate the further study on the mechanism of pathogenic fungi CYP51 and pesticide molecules, and will provide a new idea for efficient design and development of new anti-fungal drugs.

DNA-Protein Crosslink Induced by Formaldehyde and Repair in Pulmonary and Renal Cells of Rats / 环境与健康杂志

Objective To explore the DNA-protein crosslink(DPC) induced by formaldehyde(FA) and the repair.Methods Three-month-old Wistar male rats were used as the experimental animals and exposed to gaseous FA at the doses of 0,0.5,1.0,3.0 mg/m3 respectively through continuous inhalation for 72 h.The cellular suspension of the lungs and kidneys was exposed to FA at the doses of 0,25,50,100,150 and 200 ?mol/L respectively for 1 h.The KCl-SDS assay was used to detect the coefficient of DPC in the lung and renal cells.The repair at 0,6,12,18 and 24 h exposure points was determined in the exposure test of 3.0 mg/m3 for 72 h in vivo and 75 ?mol/L for 1 h in vitro.Results Higher gaseous FA(≥1.0 mg/m3) could significantly cause DPC(P

Separation and purification of the active molluscicidal component from Reineckea carnea / 中国血吸虫病防治杂志

Objective To get a molluscicide from Reineckea carnea which is effective on Oncomelania hupensis but has low poison on fish. Methods Methyl soaking, column chromatography and HPLC were used for isolation of molluscicidal component from Reineckea carnea. Results The snail mortality with Reineckea carnea powder was 86.0% for 72 h immersion and 100.0% for 120 h at the concentration of 17.5 mg/L, but the fish mortality was 0 at the concentration of 130 mg/L, indicating that Reineckea carnea had different effects on the two kinds of animals . The fraction A was obtained from Reineckea carnea by methyl immersion, column chromatography. The snail mortality with A was 84.4% for 144 h at the concentration of 10 mg/L, indicating that A had high molluscicidal effect on Oncomelania hupensis. After A being isolated by HPLC, 99.98% RC-Ⅰ was obtained from it . RC-Ⅰ was determined by API2000 LC/MS and other spectrum, and it was deduced to be one of steroidal glycosides. Conclusion Reineckea carnea contains a high effective molluscicidal component.