AIM: To construct recombinant adenoviral vector carrying the LEDGFp52 gene by homologous recombination in bacteria and to detect its expression in vitro.METHODS: The LEDGFp52 gene was cloned to adenoviral shuttle plasmid pAdTrack-CMV. Then, the resultant pAdTrack-CMV-LEDGFp52 was cotransfected into BJ5 183 bacteria with the adenoviral backbone plasmid pAdeasy-1. The adenoviral plasmid carrying LEDGFp52 was generated with homologous recombination in bacteria, and the adenoviruses were produced in 293 cells. These 293 cells were then infected with adenoviruses, and the expression of LEDGFp52 was detected by CPE (cytopathic effect) and western blot.RESULTS: The titer of Ad-LEDGFp52 adenoviruses was up to 5×1012 pfu/L after proliferation in 293 cells. LEDGFp52 was expressed efficiently in 293 cells after infection.CONCLUSION: The recombinant adenoviruses vector expressing LEDGFp52 was constructed successfully and can be used in further gene transfection experiments.

AIM: To construct and identify LEDGFp52 eukaryotic expression vector for RNA interference.METHODS: Recombinants were designed and established by targeting gene LEDGFp52 and plasmid pGensil-1 based on LEDGFp52 cDNA sequences of Genomes. Two pairs of oligonucleotides were synthesized according to the Tuschl principle and inserted into plasmid pGenSil-I to generate siRNA eukaryotic expression vector. DH5α strains were transformed, plasmids were extracted, and recombinant vectors were identified by the restriction map and the sequence analysis. The cultured cells were transfected by the recombinant plasmid (pGensil-1-RNA. LEDGFp52-1). At 48 hours after transfection, the whole cell protein was extracted, and the protein level was detected using Western blotting with mouse anti-human LEDGFp52 monoclonal antibody.RESULTS: Recombinant plasmids completely concord with the designs by the restriction map and the sequence analysis,the proteinlevel of LEDGFp52 was down regulated at 48hours after transfecting pGensil-1- LEDGFp52-1 expression vector into HeLa cells, the recombinant eukaryotic expression vectors were successfully constructed.CONCLUSION: siRNA recombinant can be successfully constructed by RNAi technique to inhibit the expression of LEDGFp52.

AIM: To construct the prokaryotic expression vector of rhLEDGFp52 gene,to obtain the rhLEDGF p52 protein.METHODS: rhLEDGFp52 gene was constructed into a prokaryotic expression vector pET30a (+) by recombinant DNA techniques and was identified by enzymatic digestion and sequence analysis. rhLEDGFp52 protein was induced expression by IPTG in E.coli BL21 (DE3), it was tested by Western blot and was purified by Ni-NTA His. Bind. Resin.RESULTS: We Successfully constructed the prokaryotic expression vector of rhLEDGFp52 gene and obtained its expression in E.coli BL21 (DE3),it was expressed in a soluble form and detected up to 34.63% of the total bacterial protein expressed in E.coli BL21 (DE3).Western blot analysis demonstrated that rhLEDGFp52 protein could spicifically integrate with LEDGF-ab. After purified by Ni-NTA His. Bind. Resin, the ultimate concentration of purified rhLEDGFp52 protein was 520μ g/ml and its purity was 87.93%.CONCLUSIONS: rhLEDGF p52 protein was obtained that provides an experimental basis for the further study of the biological function of rhLEDGFp52 protein.

ObjectiveTo compare costs of combined intravenous and inhalation anesthesia (CIIA) and total intravenous anesthesia (TIA) when lower limbs orthopedics (LLO) performed on cerebral palsy children (CPC).MethodsTo analyze data of 49 CPC who received LLO under CIIA and TIA,and compare costs of two anesthetic styles.ResultsTIA had a significantly lower cost than CIIA (P<0.001),and showed a lower incidence of post-operation nausea and vomiting.ConclusionTIA can decrease cost of anesthesia and post-operation nursing compared with CIIA.

Animals ; Hyperbaric Oxygenation ; Inflammation ; Interferon-gamma ; metabolism ; Interleukin-6 ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; therapy ; Tumor Necrosis Factor-alpha ; metabolism

Adolescent ; Adult ; Cardiomyopathy, Hypertrophic ; genetics ; Child ; Humans ; Male

Acupuncture Points ; Acupuncture Therapy ; Adult ; Combined Modality Therapy ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Melanosis ; drug therapy ; therapy ; Middle Aged ; Young Adult

Child ; Child, Preschool ; Coronary Vessels ; pathology ; Female ; Follow-Up Studies ; Humans ; Infant ; Male ; Mucocutaneous Lymph Node Syndrome ; diagnosis ; pathology ; therapy ; Retrospective Studies

OBJECTIVE:To establish an approach to determination of ketoconazole and desamethasone acetate in novel ketoconazole spraying film.METHODS:Chromatography was performed using a Waters Symmetry C 18 .The mobile phase consisted of methyl-monopotassium phosphate(4∶1).Detection wave was at239nm.The flowing rate was0.6ml/min.RESUL_ TS:Excellent linear correlations were showed when the concentrations of ketoconazole and desamethasone acetate were in the ranges of0.2～2mg/ml and0.02～0.2mg/ml respectively.The mean recoveries of ketoconazole and desamethasone acetate were98.41%(RSD=2.29%,n=6)and97.84%(RSD=2.60%,n=6)respectively.CONCLUSION:The established approach is precise,reliable and therefore applicable to the quality control of novel ketoconazole spraying film.

Objective To investigate the correlation between angiotensin converting enzyme(ACE) gene polymorphism and kawasaki disease(KD).Methods A 287 bp Alu fragment in intron 16 of the ACE gene was used as insertion(I)/deletion(D) polymorphism marker. The ACE genotype of 28 children (10 children complicated coronary dilataltion) with KD and 35 healthy controls were detected by polymerase chain reaction (PCR), and ACE concentration in blood serum was measured by ultraviolet-spectrophotometer assay.Results 1.The ACE concentration was significantly higher in KD group than that in healthy control group(P