Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris / 生物工程学报

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

Diagnosis of congenital dysfibrinogenemia / 中华检验医学杂志

Congenital dysfibrinogenemia (CD) is a hereditary disease that causes by the mutation of fibrinogen (Fg) gene, which result in abnormal of fibrinogen structure and function.Most of the mutations are dominant heredity which located at autosomal.The clinical manifestations of CD patients are highly diverse including asymptomatic, bleeding tendency, thrombophilia in some cases both bleeding tendency and thrombophilia coexist. As a result of highly diverse symptom the CD diagnosis mainly relies on laboratory tests. The result of coagulation test which has the best diagnostic value of CD was found to be fibrinogen antigen/activity ratio (PT-der/Clauss) greater than 1.43, thrombin time (TT) prolonged, prothrombin time (PT) and activated partial thromboplastin time (APTT)normal. According to patient′s clinical manifestations and coagulation function test results, combing with family history surveys diagnosis of CD can be made. Mass spectrometry can efficiently identify the type of fibrinogen defects in CD patients. And DNA sequencing can directly locate the site of mutation in fibrinogen gene.

The transformation of microcystin-LR during tap water treatment process and analysis of its degradation products / 中华预防医学杂志

Objective@#To establish a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of microcystin-LR (MC-LR) in drinking water, investigate its removal efficiency during tap water advanced treatment process and analyze its degradation products in the tap water.@*Methods@#Two parallel water samples were collected from each point of tap water advanced treatment process in September 2015, November 2015 and January 2016, respectively, and treated by mixing, filtration, concentration, elution, nitrogen blow and re-dissolvement. The samples were analyzed by LC/MS/MS to determine the MC-LR concentration and its removal efficiency during treatment process. The combination of actual water enrichment (including source water enrichment of 50 times and 1 500 times concentrated, finished water enrichment of 50 times and 2 500 times concentrated) and laboratory simulated water (including the mixture of MC-LR and liquid chlorine in the mass ratio of 1∶10, 1∶20, 1∶100 and 1∶1 000, respectively) were used to qualitative analyze the MC-LR degradation products by Orbitrap mass spectrometry.@*Results@#The linearity of MC-LR ranged from 2 to 200 μg/L with the detection limit of 0.007 9 μg/L and the limit of quantification of 0.026 3 μg/L. The recovery rate of MC-LR from different contration in drinking water were from 94.88%-101.47%. The intra-day precision was 2.51%-7.93% and the intra-day precision was 3.24%-8.41%. The average concentration of MC-LR in source water was (0.631±0.262) μg/L, 94.0% of which can be removed by ozone exposure while the concentrate was (0.038±0.016) μg/L, biological pre-treatment and chlorination. The remaining can hardly be removed by sand filtration, ozone exposure, activated carbon, ultrafiltration and other processes. The MC-LR average concentration in the finished water maintained at about (0.036±0.016) μg/L. Degradation products including hydroxy-microcystin, methyl-hydroxy-microcystin, methyl-microcystin were identified in the laboratory simulated water of the mixture of MC-LR and liquid chlorine in the mass ratio of 1∶10.@*Conclusion@#The established MC-LR detection method can be well applied to the monitoring of MC-LR in drinking water due to its simple pre-treatment process and good methodological validation parameters. The degradation products of treatment processes was different.