Expression of recombinant h-FGF21 in periplasmic space of Escherichia coli / 药学学报

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator of glucose and lipid, which is safe, effective and independent on insulin. FGF21 is considered as a prospective anti-diabetic drug. The aim of this study was to express recombinant h-FGF21 in periplasmic space of Escherichia coli. The pET27b plasmid was used to create the expression vectors of h-FGF21 with a PelB secretion signal. The ph-FGF21 (periplasmic expression of h-FGF21) was successfully expressed in the periplasm of E. coli BL21 (DE3), and soluble ph-FGF21 was isolated by disruption of the outer membrane. After twice of ion exchange chromatography, the purity of ph-FGF21 was above 95% in an analysis with a gray analysis software. The molecular weight of ph-FGF21 was about 20 kDa in SDS-PAGE and Western blotting analysis. The activity of ph-FGF21 and ih-FGF21 (intracellular expression of h-FGF21) was observed in vitro in the glucose uptake assay in HepG2 cells. The activity was observed in type 2 diabetic db/db mice after short or long-term treatments. The results suggest that the ph-FGF21 has a consistent activity with ih-FGF21 in vitro and in vivo.

The synergistic effect of FGF-21 and insulin on regulating glucose metabolism and its mechanism / 药学学报

Previous studies proposed that the synergistic effect of fibroblast growth factor-21 (FGF-21) and insulin may be due to the improvement of insulin sensitivity by FGF-21. However, there is no experimental evidence to support this. This study was designed to elucidate the mechanism of synergistic effect of FGF-21 and insulin in the regulation of glucose metabolism. The synergistic effect of FGF-21 and insulin on regulating glucose metabolism was demonstrated by investigating the glucose absorption rate by insulin resistance HepG2 cell model and the blood glucose chances in type 2 diabetic db/db mice after treatments with different concentrations of FGF-21 or/and insulin; The synergistic metabolism was revealed through detecting GLUT1 and GLUT4 transcription levels in the liver by real-time PCR method. The experimental results showed that FGF-21 and insulin have a synergistic effect on the regulation of glucose metabolism. The results of real-time PCR showed that the effective dose of FGF-21 could up-regulate the transcription level of GLUT1 in a dose-dependent manner, but had no effect on the transcription level of GLUT4. Insulin (4 u) alone could up-regulate the transcription level of GLUT4, yet had no effect on that of GLUT1. Ineffective dose 0.1 mg kg(-1) FGF-21 alone could not change the transcription level of GLUT1 or GLUT4. However, when the ineffective dose 0.1 mg x kg(-1) FGF-21 was used in combination with insulin (4 u) significantly increased the transcription levels of both GLUT1 and GLUT4, the transcription level of GLUT1 was similar to that treated with 5 time concentration of FGF-21 alone; the transcription level of GLUT4 is higher than that treated with insulin (4 u) alone. In summary, in the presence of FGF-21, insulin increases the sensitivity of FGF-21 through enhancing GLUT1 transcription. Vice versa, FGF-21 increases the sensitivity of insulin by stimulating GLUT4 transcription in the presence of insulin. FGF-21 and insulin exert a synergistic effect on glucose metabolism through mutual sensitization.

Preparation and characterization of human phage display antibody against peroxiredoxin I of lung adenocarcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a human phage antibody library and screen the single chain variable fragment (ScFv) antibudies to peroxiredoxin I (Prx I) of lung adenocarcinoma.</p><p><b>METHODS</b>The total RNA was isolated from the lymph nodes of lung cancer patients to amplify V(H) and V(L) genes by RT-PCR. V(H) and V(L) were linked with a DNA linker by SOE-PCR to construct the single chain variable fragment gene. The ScFvs were coloned into the phage vector pCANTAB5E. The insert ratio of the ScFv antibody library was identified by PCR, and the products were digested by SfiI/NotI and analyzed with 1% agarose gel electrophoresis. Three rounds of panning against lung adenocarcinoma cell line A549 and Prx I were performed, and the positive clones were identified for soluble expression. The soluble antibodies were identified by SDS-PAGE and Western blotting, and ELISA and immunocytochemistry were used to characterize the activity of the antibodies.</p><p><b>RESULTS</b>A recombination phage antibody library was constructed. The insert ratio of ScFv gene was 77% (23/30), and enzyme digestion identified the target product. The sixth phage harvest resulted in a yield 180 folds of that of the first one. Positive reactions to A549 cells were detected in 6 of 10 random clones, with a positivity rate of 60%. The soluble human ScFvs against Prx I of lung adenocarcinoma were expressed in E. coli HB2151 and confirmed by SDS-PAGE and Western blotting. ELISA and immunocytochemistry demonstrated a relative specific affinity of the soluble antibodies to A549 cells.</p><p><b>CONCLUSION</b>ScFv antibodies against lung adenocarcinoma have been acquired by phage display antibody library technique, and the soluble antibodies have a relative avidity specific to human lung adenocarcinoma A549 cells overexpressing PrxI.</p>

Clinical characteristics of 115 Chinese patients with antiphospholipid syndrome and the analysis of different classification criteria / 中华风湿病学杂志

Objective To analyze three different classification criteria, the clinical characteristics of antiphospholipid syndrome(APS)in a cohort of Chinese patients. Methods From January 1996 to October 2006, APS patients diagnosed with different classification criteria were retrospectively studied. Results There were totally 120 APS patients fulfilled at least one criterion, One hundred and one patients fulfilled the 1988 Asherson criteria, 96 patients fulfilled the 1999 Sapporo criteria, and 115 patients fulfilled the 2006 Sydney criteria. The ratio of male to female in a cohort of 115 definite APS patients was 1 to 10.5. The mean period of the disease until entry into the study was 82.6 months, the mean age at study entry was(41?12)years. Ninety patients had thrombosis episodes, among which the most common presenting manifestations were deep venous thrombosis, stroke and skin vasculitis. Forty-six of 92 married women in our cohort had fetal morbidity. Catas- trophic APS occurred in 7 patients. The presence of anticardiolipin antibodies(aCL)was detected in 86 pa- tients, anti-beta-2 glycoproteinⅠantibodies in 58 patients and lupus anticoagulant(LA)in 27 patients. Conclusion The most common presenting manifestations are deep venous thrombosis, stroke and cutaneous manifestations. The sensitivity of Sydney classification criteria is improved by adding anti-beta-2 glycopreteinⅠantibody as one of the laboratory criteria. However, primary APS patients who only presented with thrombo- cytupenia and positive laboratory tests could not satisfy this criterion. In addition, the significance of autoanti- bodies to some coagulant factors in APS needs further study.