Screening and identification of auto-antigen RHDAG1 of rheumatic heart disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the candidate auto-antigen of rheumatic heart disease as a molecular marker for this disease.</p><p><b>METHODS</b>The total RNA of the heart tissue of patients with rheumatic heart disease was extracted and reverse-transcribed into long cDNA to construct the phage expression library. The library was screened using the serum from patients with active rheumatic fever, and the positive clone was identified and analyzed by bioinformatics and expressed in vitro. The expressed products were evaluated with Western blotting and its cross-reactivity was assessed.</p><p><b>RESULTS</b>The phage expression library of the heart tissue of patients with rheumatic heart disease was constructed, with the titer of the primary library of 3.3×10(6) pfu/ml, recombinant rate of 99%, and 81% of the inserted segments were larger than 1 kb. An auto-antigen RHDAG1 was identified by screening, which was homologous to keratin 18. RHDAG1 was detected in the serum of patients with active rheumatic fever and of those with rheumatic heart disease, but not in the serum of healthy subjects.</p><p><b>CONCLUSION</b>Phage display library can be an effective strategy to screen the auto-antigens of rheumatic heart disease. The auto-antigen RHDAG1 can be a candidate molecular biomarker of rheumatic heart disease and/or rheumatic fever.</p>

Identification and tissue localization of intermediate filament protein in Angiostrongylus cantonensis / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the type of the intermediate filament (IF) protein of Angiostrongylus cantonensis and analyze its tissue localization.</p><p><b>METHODS</b>Recombinant pET-IF of antigen IF was expressed in E.coli with IPTG induction, and the expression products were purified by His.Bind column and identified for determining the type of the IF protein by Western blotting. Anti-IF antibody was prepared by multi-spot subcutaneous injection into mouse and used to detect the tissue slices of A. cantonensis by immunohistochemical analysis.</p><p><b>RESULTS</b>The antigen IF were correctly expressed and purified, and identified as a keratin located in the intestine wall and cytoplusma.</p><p><b>CONCLUSION</b>The antigen IF is distributed in the intestine wall of A. cantonensis.</p>

Induced differentiation and signaling factor PTEN expression of 3T3-L1 adipocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To optimize the condition for inducing the differentiation of 3T3-L1 preadipocytes into adipocytes and study the expression of PTEN tumor suppression gene in this process, aiming to understand the regulatory role of PTEN in normal adipocyte differentiation and collect laboratory evidence for developing drugs targeting PTEN.</p><p><b>METHODS</b>The differentiation of 3T3-L1 preadipocytes cultured in high-glucose DMEM were induced according to 2 protocols with different combinations of dexamethasone, isobutylmethylxanthine (IBMX) and insulin, and the resultant adipocytes were identified by oil red O staining. The total proteins of 3T3-L1 were extracted and analyzed by Western blotting, and PTEN homology between mice and human was analyzed by bioinformatic method.</p><p><b>RESULTS</b>For optimized 3T3-L1 differentiation, 3T3-L1 cells were initially induced with the combination of 1 micromol/L dexamethasone, 0.5 mmol/L IBMX and 5 microg/ml insulin for 48 h, followed by treatment with 5 microg/ml insulin in 4.5 g/L glucose DMEM for 48 h, which resulted in high differentiation rate of 3T3-L1 cells (up to 90% on the 10th day) with unified morphology and size. PTEN expression varied quantitatively in the process of differentiation, especially low on the 12th day as compared with those measured on days 4, 6 and 9. The mice PTEN mRNA shared 96% homology and PTEN amino acid 100% homology with their human counterparts.</p><p><b>CONCLUSION</b>Endogenous PTEN expression is down-regulated during 3T3-L1 differentiation, suggesting that PTEN may enhance insulin sensitivity and promote adipogenesis under physiological conditions.</p>