Objective To express human single-chain antibody against amyloid?peptide 40 in pichia pastoris for passive immuniza- tion to Alzheimer's disease patients.Methods Human single-chain antibody gene from a phage display library was mutated to obliterate BamHI site and cloned into pAO815 plasmid for pT-scFvA?40 construction which was identified by PCR amplification and endonuclease digestion.Vector pT-scFvA?40 was cut by EcoRI and NotI endonucleases and the antibody gene was cloned into pPICgK plasmid to con- struct pPIC9K-scFvA?40 expression vector which was confirmed by gene sequencing.Linearized pPIC9K-scFvA?40 was used to trans- form pichia pastoris GS115 and the recombinant was induced by 0.5% methanol to express human single - chain antibody against amyloid?peptide 40.Results Gene sequencing confirmed pPIC9K-scFvA?40 orientation.Recombinants were obtained by linearized pPICgK - scFvA?40 transformation.After inducing with 0.5% methanol,the recombinant secreted proteins with 33 kD size.Conclusions Expression vector pPIC9K-scFvA?40 was constructed successfully.Human single-chain antibody against amyloid?peptide 40 was recombinantly expressed in pichia pastoris.

Objective To investigate the relationship between cysteinyl leukotriene and clinical response to antileukotriene treatment,and to help select a clinical pharmacologic scheme.Methods Seventy-eight cases with acute mild-moderate asthma were treated with montelukast in a four-week trial.Asthmatic symptom score,usage of ?_2 receptor agonist,percentage of eosinophil,serumal IgE concentration,spirometry and urinary leukotriene E_4(uLTE_4)were measured pre- and post-treatment.Logistic analysis was used to access the various clinical parameters correlated with the response to montelukast.Results There were 48 responders and 30 nonresponders.The uLTE_4 level from the responders was higher than that of nonresponders(P0.05).Subjects with a uLTE_4 level of ≥1 200 pg/mL were11.5 times more likely to respond to montelukast than those with

Objective To prepare a novel annexin V and conjugate it with fluorescein isothiocyanate and test their binding ability to phosphatidylserine(PS)exposed erythrocytes.Methods The annexin V fused with an metal chelating binding site was obtained from pichia pastoris culturing and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ion-exchange chromatography and ammonium sulfate precipitation.The annexin V was conjugated with fluorescein isothiocyanate in boric buffer and the conjugate annexin V-FITC was purified by ion-exchange chromatography.Sheep red blood cells were treated with Glutaraldehyde to expose membrane PS.The annexin V-FITC binding to PS exposed erythrocytes was investigated by fluorescent microscopy.Results The novel annexin V was purified and concentrated from pichia pastoris culture by ion-exchange chromatography and ammonium sulfate precipitation.After conjugating with FITC,the annexin V was found to bind PS exposed erythrocytes by fluorescent microscopy.Conclusion The annexin V expressed recombinantly in pichia pastoris retains the binding ability to PS exposed erythrocytes and is applicable to apoptosis detection in vitro.

Objective To prepare human annexin V dimmer and make chance for its pharmacological research.Methods Pichia pastoris transformed by human annexin V gene was used in this investigation.The engineered yeast was cultured in BMGY media and induced with methanol media BMMY for secreting expression of human annexin V.The final BMMY media supernatant was adopted for preparation of human annexin V dimmer by ammonium sulfate precipitation,molecular sieve separation and ultra-filtration concentration.The purification process was monitored by SDS-PAGE and coomassie brilliant blue staining.Results Human annexin V was obtained after two days induction of pichia pastoris in BMMY media.The human annexin V dimmer was successfully prepared from culture supernant by serial ammonium sulfate precipitation,molecular sieve separation and ultra-filtration concentration.Conclusion We prepare the human annexin V dimmer(diannexin V)successfully.

Objective To derermine the affinity of site-specific labeled annexin V with ~(99m)Tc to phosphatidylserine (PS)exposed erythrocytes.Methods The annexin V fused with a metal chelating binding site was obtained from Pichia Pastoris culture and methanol induction expression.The annexin V was purified from the culture supernatant crude product by uhrafihration.The annexin V was conjugated with ~(99m)Tc site-specifically through sodium glucoheptonic acid and SnCl_2.The radioactive annexin V was added to determine its affinity to PS exposed erythrocytes.Results The calcium concentration at which half of the protein is bound to cells(EC50)is changed with varying ratio of protein to cells.The affinity was determined at a low protein/cell ratio as 33.4.Conclusion The annexin V recombinantly expressed in Pichia Pastoris shows high affinity to PS exposed erythrocytes.

A questionnaire survey on chronic heart failure management was conducted in 110 physicians from Xindu,Supo,Jiajiang and Xinhua community hospital in Chengdu from January 2007 to June 2010.Results showed that 77.3％ (85/110) of community physicians lacked knowledge about prevention,diagnosis and treatment of chronic heart failure,and the diagnostic accuracy rate was only 51.3％ (40/78) in these community hospitals.There was lack of awareness of the guideline of chronic heart failure:the rate of β blockers use was 15.0％ (6/40) and only 5.0％ (2/40) used the target dose; the rate of angiotensin converting enzyme inhibitor use was 17.5％ (7/40) and only 7.50％ (3/40) used the target dose.In addition,90％ (99/110) of community doctors lacked the education and management for patients with heart failure.The survey suggests that the current situation of chronic heart failure management in community physicians in Chengdu is unsatisfactory.It is necessary to strengthen the training of community physicians.

Objective To investigate the suppression effect of expressing parvovirus H-1 nonstructural protein 1 (NS1) gene on human gastric cancer cells and the possible mechanisms.Methods A recombinant enhanced green fluorescent protein (eGFP) labeled NS1 of parvovirus H-1 plasmid was constructed.Human gastric cancer cell line SGC7901 was transfected with recombinant plasmid (experiment group) or blank vector (negative control group) and blank control group was treated with equal amount of phosphate buffered saline (blank control group).After transfection,the distribution of fluorescent signal was observed under fluorescent microscope.The expression of NS1 at gene and protein level was measured.Cell growth curve of each group was drawn.The expression of cell senescence-associated β-galactosidase (SA-β-Gal) was tested.The changes of cell cycle were investigated by flowcytometry.Two groups' comparision was performed by t-test.Results After transfection,NS1 was expressed in SGC7901 cells at gene and protein level.Compared with negative control group,the fluorescent signal accumulated in cell nucleus in experiment group.The percentage of SA-β-Gal positive cell in experiment group ((30.5 ± 1.4) ％) was higher than that of negative control group ((4.4± 1.1) ％) and the difference was statistically significant (t =-12.931,P ＜ 0.01).The growth inhibition rate of SGC7901 cells from the first day to the fourth day was 45％,62％,73％ and 77％,respectively.The cell cycle of eGFP-NS1 expressed SGC7901 cells was arrested at G0/G1 phase.Conclusion Parvovirus H-1 NS1 play the role in cell nucleus of gastric cancer cell line SGC7901 and could make cell cycle arrested at G0/G1 phase,which effectively inhibited the proliferation SGC7901 cell.

The microflora plays an important role in providing nutrients to the intestinal mucosal cells.The disruption of the intestinal microflora balance may modify the intestinal barrier function, and influence the health.Dietary fibre can be divided into solububle fibre and insoluable fibre.Insoluable fibre plays an important role in faecal bulking and prevention of constipation.The human body can obtain energy for growth and maintenance of cellular function from fermentation of solable fibre.

Objective:To construct an adenovirus-mediated mouse endostatin vector (Ad-mEndo) and to observe its inhibitory effect on the pulmonary metastasis of osteosarcoma in nude mice, so as to discuss the relationship between ES expression and the pulmonary metastasis of osteosarcoma. Methods: Recombinant adenovirus plasmid pDC315-mEndo was constructed and used to prepare recombinant Ad-mEndo. Osteosarcoma MG-63 cells were subcutaneously injected into the right fore limbs to establish nude mouse model of osteosarcoma; and the models were randomly divided into 3 groups: Ad-mEndo group, Ad-EGFP group and PBS group; animals receiving no transplantation served as blank control. The corresponding agents were injected (20 ?l per time) for a consecutive of 5 times on a weekly basis. The tumor volumes, histopathological characteristics were observed; ELISA was employed to examine the serum ES level. Animals were sacrificed 7 weeks later and the pulmonary metastasis was observed. Results: Sixteen days later,the tumor volume was (1.53?0.05)cm3 in Ad-EGFP group, (1.56?0.07)cm3 in the PBS group, and (0.91?0.03)cm3 in the Ad-mEndo group, with the tumor inhibitory rate being 40.7% in the last group. The serum ES level in the Ad-mEndo group was significantly higher than that of the other groups (P

Objective: The influence on the blood glucose,ALB,LYM,K+,Na+,Cl-,TG,TC and so on after the use of different nutrition formula was observed in patients with cerebrovascular disease and type 2 diabetes.Methods: 40 patients who suffered from cerebrovascular diseases and type 2 diabetes were divided into two groups: one group using the ordinary formula and the others using special-purpose formula of diabetes.The levels of blood glucose,ALB,LYM,K+,Na+,Cl-,TG,TC were monitored.Results: The patient's blood glucose after use of ordinary formula rised obviously and insulin or the oral drug for decreasing blood glucose was used.The blood glucose in special-purpose diabetes formula group didn' t increase obviously,and there was not the need of using insulin(P