Objective To construct the recombinant plasmid pET-28a-CV2025 by using the transaminase(CV2025)gene sequence of Chromobacterium violaceum and express it in Escherichia coli(E.coli).Methods The recombinant plasmid pET-28a-CV2025 was constructed by linking the gene sequence of CV2025 with pET-28a(+)vector,which was transformed into E.coli TOP10 for screening of positive clones. After double enzyme digestion and sequencing verification,the recombinant plasmid was transformed into E.coli BL21(DE3)and Rosetta,and was induced to express the target protein. The recombinant strain 28a-CV2025-BL21 was activated and inoculated into 2TY liquid medium. After IPTG induced expression,with isopropylamine as the amino donor and 1-（4-methoxyphenyl）acetophenone,o-fluoroacetophenone and acetophenone as the amino receptor,the reaction was monitored by thin layer chromatography,and the transaminase activity was preliminarily tested. Finally,the reaction was further identified and analyzed by high-performance liquid chromatography.Results The recombinant plasmid pET-28a-CV2025 was constructed correctly as identified by double enzyme digestion and sequen-cing. The expression products of 28a-CV2025-BL21 and 28a-CV2025-Rosetta showed target protein bands with the relative molecular mass of about 51 000,and most of the proteins expressed by 28a-CV2025-BL21 existed in soluble form. The enzyme solution catalyzed the transamination reaction of 1-（4-methoxyphenyl）acetophenone with isopropylamine to generate the corresponding chiral amine 1-（4-methoxyphenyl）ethylamine,which had certain catalytic activity.Conclusion In this study,CV2025 was successfully expressed in E.coli,and the soluble protein in the supernatant was preliminarily tested to have catalytic activity,which lays a foundation for the later separation and purification of transaminase and the enhancement of product conversion rate

Adenocarcinoma ; diagnosis ; drug therapy ; genetics ; metabolism ; Antineoplastic Agents ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; diagnosis ; drug therapy ; genetics ; metabolism ; Carcinoma, Squamous Cell ; diagnosis ; drug therapy ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Discoidin Domain Receptors ; Glutamates ; therapeutic use ; Guanine ; analogs & derivatives ; therapeutic use ; Humans ; Lung Neoplasms ; diagnosis ; drug therapy ; genetics ; metabolism ; Molecular Diagnostic Techniques ; methods ; Mutation ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Pemetrexed ; Protein Kinase Inhibitors ; therapeutic use ; Proto-Oncogene Proteins B-raf ; genetics ; metabolism ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; genetics ; metabolism ; Receptors, Mitogen ; genetics ; metabolism ; Transcription Factors

OBJECTIVE:To improve the effectiveness of experimental teaching and independent hands-on ability of the students.METHODS:Combined with teaching characteristics of newly established universities,the characteristics of instrumental analytical experiment teaching and the characteristics of pharmaceutical students,flipped classroom and project modular design were applied to instrumental analytical experiment teaching together.The instrumental analytical experiment teaching was expounded and integrated in respects of curriculum content design,teaching implementation and curriculum assessment.RESULTS&CONCLUSIONS:The students achieve the knowledge learning after classroom,and complete the knowledge absorption and mastering in class with the help of video and audio information.It also improved independent learning ability of the students,strengthened the ability of interactive learning and cooperative leafing between teachers and students and among students.It is feasible to apply the flipped classroom and the experimental project modular design in the instrument analytical chemistry experiment teaching reform.This method also can provide some references for the reform of instrument analytical chemistry experiment teaching.

Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein,infected-cell protein 22 (ICP22), in the nucleus by observing the localization of ICP22-EGFP fusion protein.Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus,persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracyclinedependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of internal ribosome entry sites (IRES) on transcriptional regulation.

Objective To evaluate the effects and side effects of three different recruitment maneuvers (RM) in acute respiratory distress syndrome (ARDS) caused by extrapulmonary disease.Methods Forty-four patients of extrapulmonary ARDS, according to crossover design methods, were undergone three RM in different periods, including sustained inflation (SI), increase progressively positive end expiratory pressure (IP), pressure control ventilation (PCV). Heart rate (HR), mean arterial blood pressure (MAP), central venous pressure (CVP), oxygenation index, lung compliance were recorded before and after RM, and were analyzed for statistical analysis. Results Oxygenation index and lung compliance were increased obviously in a short time after RM, improvement of IP method were better more obviously than the other two methods [1 h oxygenation index after RM:227 ± 42 vs 190 ± 19,186 ± 21; lung compliance:(59.4±12.5 ) ml/cm H2O(1 cm H2O = 0.098 kPa) vs (50.1 ± 9.3 ), (49.7 ± 10.6) ml/cm H2O;P＜ 0.05], 2h after RM,there were no statistical difference among the three methods (P＞0.05). After RM,HR and CVPwere increased, MAP was decreased in a short time, changes of SI method were smaller than the other two inethods [10 min HR after RM: (94.0±10.3 ) beats/min vs (116.0 ± 14.8 ), ( 107.0 ± 5.7 ) beats/min; CVP:(13.7±3.1 )cm H2O vs ( 18.4 ± 6.7 ), ( 15.4 ± 2.7 )cm H2O; MAP: ( 87.0 ± 12.1 ) mm Hg( 1 mm Hg = 0.133 kPa) vs (73.0 ± 4.8), (81.0 ± 6.6) mm Hg;P＜ 0.05), 20 min after RM, there were no statistical difference among the three methods (P＞ 0.05). Conclusion When extrapulmonary ARDS undergo RM ,IP method is the most effective on increasing oxygenation index and lung compliance, SI method has the smallest side effect on hemodynamics.

Objective To study the effects of semaphorin 5A (SEMA5A) gene silencing by lentivirus?mediated short hairpin RNA(shRNA)on biological activity of malignant melanoma cell line A375. Methods Two pairs of interference sequences for SEMA5A gene(shRNA1 and A375?shRNA2)and a pair of control interference sequences were designed to build lentiviral vectors, which were then transfected into HEK293T cells to gain lentivirus. A375 cells were divided into three groups:experimental group(A375?shRNA1 and A375?shRNA2 cells)transfected with the lentivirus containing shRNA1 or shRNA2, negative control group (A375?con cells) transfected with that containing the control shRNA, and blank control group(A375 cells)receiving no transfection. The A375 cells with stable knockdown of SEMA5A gene expression were screened by puromycin. Subsequently, reverse transcription?PCR and Western?blot analysis were performed to detect mRNA and protein expressions of Semaphorin 5A in these cells, and methyl thiazolyl tetrazolium(MTT)assay was applied to evaluate the growth of cells. The scratch assay and invasion assay were conducted to estimate migration and invasion ability of cells. Results The lentivirus containing the SEMA5A?targeting shRNAs or control shRNA was successfully transfected into A375 cells, and stably transfected cells were gained after puromycin selection. The expressions of semaphorin 5A mRNA and protein in the A375?shRNA2 cells were significantly reduced compared with those in the A375?con and A375 cells(all P < 0.05). MTT assay showed that the growth of A375?shRNA2 cells was significantly slower than that of A375?con and A375 cells(both P<0.05), while there was no significant difference in the growth rate between A375?con and A375 cells(P>0.05). The scratch assay showed that there was no obvious cell migration into the scratch in the experiment group, whereas the scratch was almost covered by cells in the negative control group and blank control group. The invasion assay showed that the number of A375?shRNA2 cells passing through the Transwell chamber was significantly smaller than that of A375 and A375?con cells(both P<0.05), while there was no significant difference between that of A375 and A375?con cells(P > 0.05). Conclusion The silencing of SEMA5A gene by lentivirus?mediated shRNA could effectively down?regulate the expression of semaphorin 5A, and inhibit the growth, invasion and migration of A375 cells.

Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years,the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. A novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR),and point mutations including M41L,K70R,K103N,Y181C,T215F were detected. A longer mutant type primer was designed,using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity,low cost,relatively less time consumption and high-throughput screening. It will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.

HIV-1 envelope glycoprotein gp41,which is a hopeful target for HIV-1 fusion inhibitors,plays a critical role in the fusion of viral and cellular membranes.In order to build up the screening assay of HIV-1 fusion inhibitors targeting gp41,HIV-1 gp41 5-helix and 6-helix were expressed in prokaryotic cells.Gp41 5-helix and 6-helix recombined plasmids were constructed by using PCR,enzyme digestion and ligation taking the clade B HIV-1 genome as a template.The plasmid was transferred into E.coli BL21(DE3)and then induced by IPTG.The expressed protein was purified by affinity chromatography after denaturation and renaturation.The SDS-PAGE analysis was used during expression and purification.Native-PAGE was used to identify the interaction between gp41 5-helix and T-20.The result will be helpful to build up the screening assay of HIV-1 fusion inhibitors targeting gp41.

Objective To develop a safe,practical,disposable slide culture medium for fungi culture.Methods Based on the principle of traditional slide culture medium with small steel ring,a plate of polyvinyl chloride (PVC) with high transparency was chosen as the bottom material of the new medium which held the size of the conventional slide (25.2 mm × 75.9 mm × 1.05 mm) with frosted design at both ends.The diameter of inoculation hole was 2.4 mm on the rounded culture dish which was designed with diameter of 19.4 mm and side height of 3.75 mm.The users could inject potato dextrose agar (PDA) or other medium into the culture dish as needed and then sealed it with a plug.The upper cover was prepared with toughened glass.The improved disposable slide culture medium should be kept in humidifying box with sponge strips in the water tanks of both sides and sealed with cap.The growth status,microscopic morphology and staining result of filamentous fungi in the totally enclosed slide culture medium were observed and the preservative status of the prepared medium was also monitored simultaneously.Results The effects of the improved slide culture medium were satisfactory in clinical practical application.The growing status of fungi could be observed visually and the pigment was clear.The original growth form of fungi could be monitored under microscope and dye material could be perfused directly to stain with good results.The appearance and the volume of packaged slide medium were unchanged after preservation at 4 ℃ for 3 months.Conclusion An improved slide culture medium was successfully developed,which should be easy to operate,high visible,satisfactory for sealing effects and reliable for culture performance with high biological safety.The growth status of fungi could be observed under microscope at any time,and the medium could also be monitored under oil immersion lens directly and stained with cotton blue.The improved medium could be used in morphological examination for fungi in different levels of medical laboratories since its favorable results in clinical application.

Objective To observe effects of different extracts of Jianpi Huogu Formula (JPHGF) on proliferation and differentiation of bone marrow mesenchymal stem cell (BMSCs). Methods Whole bone marrow adherent was used to screen, culture, and isolate BMSCs. Extracts from different parts (water, chloroform, ethyl acetate and n-butanol parts) of JPHGF were administrated for a certain time. MTS was used to detent cell proliferation;ALP staining was used to detect ALP activity;ARS staining was used to detect the formation of calcium nodules;oil red O staining was used to detect fat cell formation. Results Extracts from different parts of JPHGF could promote cell proliferation of BMSCs in different levels, followed by its strength in water, chloroform, ethyl acetate, and n-butanol parts;ALP staining results showed that the intensity of ALP expression of the order is water, acetic acid ethyl, chloroform and n-butanol parts;in promoting the formation of calcium nodules, ARS staining results showed that its intensity were water, chloroform, ethyl acetate, and n-butanol parts;oil red O staining results showed that inhibition intensity of fat cells interaction strength was formed from ethyl acetate, water, chloroform to n-butanol parts. Conclusion Extracts from different parts of JPHGF have different effects on BMSCs proliferation and differentiation. Water extraction has the strongest osteogenic differentiation and proliferation, and ethyl acetate has the best effect on the inhibition of cell formation.