The partial segment of Marek′s disease virus (MDV) glycoprotein B (gB) gene was amplified by PCR. The segment was cloned into pET-28a vector to obtain the recombinant pET-gB plasmid. The recombinant plasmid was transformed into E.coli BL21,and expressed in very high level as inclusion body after induced with 1.0mmol/L IPTG. The inclusion body was solubilized in urea (8mol/L) . The purified protein was obtained by use of His?Bind affinity chromatography. Mice were immunized i.p. by the purified protein to make the polyclonal antibody. The titer of the antibody by indirect ELISA was 1?10~ -5 . Moreover, the analysis by western blot proved that antibody was specific to the recombinant protein. These works lay a favorable foundation for the study of the immune response by MDV gB.

OBJECTIVETo establish and evaluate a BA-ELISA method for the quantitative detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein.

METHODSWe deliberately selected three tables of CFTR and made the synthetic peptide be expressed in E. coli, then used the antigen to immunize rabbits to obtain the anti-CFTR polyclonal serum. After that, 96 well plates were coated with the purified antibody against CFTR. The antigen CFTR which was extracted from human sperm was detected by anti-CFTR antibody labeled with biotin, horseradish peroxidase conjugated avidin, and the substrate. The concentrations of two kinds of antibodies and the experiment parameters were optimized. Thereby, the double antibody sandwich BA-ELISA method for the quantitative detection of CFTR protein was established. Furthermore, the reproducibility, specificity and so on were evaluated by clinical specimens of sperm.

RESULTSThe optimal concentration of coated anti-CFTR IgG was 4 µg/ml, while the biotin labeled anti-CFTR IgG was 10 µg/ml; the optimal blocking buffer was 1% BSA-PBST, the optimal time of the reaction between antigen and antibody was 60 min, the optimal chromogenic time was 15 min, the intra-assay and inter-assay coefficient were 2.16%-9.23% and 2.29%-11.71% respectively; The lowest detectable limit was 0.15 ng/ml; the standard curve had a good linear correlation of R2 = 0.962.

CONCLUSIONThe BA-ELISA method for the quantitative detection of CTFR protein is successfully established, and it is demonstrated that the method has strong specificity, high sensitivity and good reproducibility. It provides the basis and evidence of the further application of the method.

Animals ; Antibodies ; Cystic Fibrosis Transmembrane Conductance Regulator ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; Humans ; Peptides ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity

The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.

Objective:To establish a method for the determination of sodion in cefalotin sodium by ion chromatography and investi-gate the salt-forming rate of the products. Methods: A TSKgelSuper IC-CR cation exchange column (150 mm × 4. 6 mm, 3. 0 μm) was used. The mobile phase was the mixture of 2. 2 mmol·L-1 methanesulfonic acid and 1 mmol·L-1 18-crown-6-ether with the flow rate of 0. 8 ml·min-1 . The column temperature was 40℃ and the injection volume was 20μl. The detector was an electric conductiv-ity detector. Results:The linear correlation of sodion was good within the range of 3. 0-60. 0μg·ml-1(r=0. 999 9). The average re-covery was 99. 8%(RSD=0. 8%, n=9). The mole number ratio of sodion to cefalotin was within the range of 0. 97-1. 03. Conclu-sion:The method is specific, precise and accurate, and can be used in the determination of sodion in cefalotin sodium. The salt-form-ing rate of the 8 batches of samples is promising.

Objective:To establish an HPLC coupled with post column derivatization method for the determination of gentamicin C components and the related substances based on the latest European Pharmacopeia and compare with the electrochemical method. Methods:A Hydrophilic C18(250 mm ×4.6 mm, 5 μm)column was used with acetonitrile-50 mmol·L-1 sodium hydroxide solution ( pH 2. 6) containing 0. 7% trifluoroacetic acid and 0. 025% pentafluoropropanoic acid (1. 5∶98. 5) as the mobile phase. The temper-ature of post-column reaction was set at 30℃, and the samples were detected by a fluorescence detector withλex of 340nm andλem of 430nm. A pulsed amperometric detector (PAD) was applied in the electrochemical method with golden working electrode in a four-po-tential working mode. Results: According to the results of the two detection methods, the linear range of C1a , C2 , C2a and C1 was 5.82-233.00,6.92-277.00,4.00-160.00and6.23-249.00 μg·ml-1(r >0.9993) , respectively. The limit of detection and quantization were 0. 92-3. 28ng and 1. 37-5. 19ng, respectively. Conclusion:There is no significant difference between the determina-tion results of the two methods.

OBJECTIVETo study the association of functional bladder capacity with the severity of bedwetting in children with nocturnal enuresis.

METHODSA questionnaire investigation was performed in 1 500 children with nocturnal enuresis and the functional bladder capacity was examined by B-ultrasound.

RESULTSThe ratio of males to females was 1.3:1. The majority of patients (87%) were in an age range of 5-10 years, followed by the 10-14 years group (12%), and the 15-18 years group (1%). Six hundred and thirty-seven patients (42.4%) showed a decreased functional bladder capacity (less than 50% of normal level). The patients were classified into four groups according to the severity of bedwetting (from severe to mild): > or =2 times per night (n=53, 3.5%), > or =7 times per week (n=969, 64.6%), 3-6 times per week (n=380, 25.3%) and 1-2 times per week (n=98, 6.5%). The incidence of the reduction in functional bladder capacity in the above four groups was 79.2%, 48.3%, 29.7% and 14.3% respectively and a significant difference was noted among the four groups.

CONCLUSIONSMost of children with nocturnal enuresis showed decreased functional bladder capacity. Functional bladder capacity is associated with the severity of bedwetting in children with nocturnal enuresis.

Child ; Child, Preschool ; Female ; Humans ; Male ; Nocturnal Enuresis ; physiopathology ; Urinary Bladder ; physiopathology

Amino Acids ; metabolism ; Animals ; Dose-Response Relationship, Radiation ; Hippocampus ; metabolism ; radiation effects ; Male ; Microwaves ; adverse effects ; Neurons ; radiation effects ; ultrastructure ; Rats ; Rats, Wistar ; Synapses ; radiation effects ; ultrastructure

OBJECTIVETo study the chemical constituents in Agrimonia pilosa.

METHODThe compounds were isolated and purified by various column chromatographic methods and elucidated on the basis of chemical and spectroscopic evidences.

RESULTNine flavonoids were obtained and identified as tiliroside (1), kaempferol 3-O-alpha-L-rhampyranoside (2), quercetin 3-O-alpha-L-rhampyranoside (3), quercetin 3-O-beta-D-glucopyranoside (4), kaempferol 3-O-beta-D-glucopyranoside (5), kaempferol (6), apigenin (7), luteolin (8), quercetin (9).

CONCLUSIONCompounds 1-3, 5, 6 and 8 were isolated from this plant for the first time.

Agrimonia ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Flavonoids ; chemistry ; isolation & purification

OBJECTIVETo investigate the expression of occludin, ZO-1, MMP-2, and MMP-9 in cerebral microvasculature following Pulse Electromagnetic Field (PEMF) induced BBB permeability change.

METHODSSprague-Dawley rats were randomized into PEMF and sham exposed groups (n = 8). After exposure to PEMF at 0.5, 1, 3, 6, and 12 h, BBB permeability was measured by Evans-Blue extravasation. The expression of occludin, ZO-1, MMP-2, and MMP-9 were detected by real-time quantitative reverse transcriptase PCR and western blotting. MMP-2 and MMP-9 activity were detected by EnzChek gelatinase assay.

RESULTSCompared with the sham group, PEMF exposure led to increased permeability of the BBB to EB, which was prolonged after exposure. BBB permeability became progressively more severe, and recovered at 6 h. The gene and protein expression of occludin and ZO-1 were significantly decreased, while MMP-2 and MMP-9 expression were significantly increased after exposure to PEMF. All levels of expression recovered 12 h following PEMF.

CONCLUSIONChanges to BBB permeability were related to the alteration expression of tight junction proteins and matrix metalloproteinase after exposure to PEMF.

Animals ; Blood-Brain Barrier ; Electromagnetic Fields ; Male ; Matrix Metalloproteinases ; metabolism ; Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tight Junctions ; metabolism

AIMTo develop a rapid and sensitive LC/MS/MS method for the analysis of levodropropizine in plasma and study the pharmacokinetics of levodropropizine in healthy Chinese volunteers.

METHODSLevodropropizine and zolmitriptan (internal standard, IS) were extracted from plasma samples and chromatographed on a C18 column and detected using a tandem mass spectrometer with a TurboIon Spray ionization interface. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions of the m/z 237 --> m/z 120 for levodropropizine and m/z 288 --> m/z 58 for the IS.

RESULTSThe limit of quantification of the method for levodropropizine was 0.25 microg x L(-1). The assay was linear over the concentration range from 0.25 to 500.0 microg x L(-1) and intra- and inter-day precision over this range were < 11.4% with good accuracy.

CONCLUSIONThe method is shown to be accurate, and suitable for clinical pharmacokinetic study of levodropropizine.

Administration, Oral ; Antitussive Agents ; blood ; pharmacokinetics ; Area Under Curve ; Chromatography, Liquid ; Humans ; Male ; Propylene Glycols ; administration & dosage ; blood ; pharmacokinetics ; Spectrometry, Mass, Electrospray Ionization