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.

At the time of phage’s discovery, phage therapy was regarded as a possible treatment method against bacterial infection. Although phage therapy was used to treat and prevent bacterial infection in the former Soviet Union and Eastern Europe, it was abandoned by the West in the 1940s with the arrival of the antibiotic era. However, the ongoing evolution of bacterial multidrug-resistance has recently motivated the Western scientific community to reevaluate phage therapy for bacterial infections that are incurable by conventional chemotherapy. With the indepth study of phages, it’s increasingly acknowledged that phages, as the medicine to cure bacterial infection, are convenient, safe and efficient therapeutics. This paper summarizes the recent years’ advanced researches in this area.

OBJECTIVETo establish a method to produce virus-like particles (VLP) of poliovirus type I in Saccharomy cescerevisiae to develop potential novel recombinant vaccine against poliovirus type 1.

METHODSThe genes of P1 and 3CD of poliovirus type I were optimized, synthesized and inserted into expression vector, which was further transfected into Saccharomy cescerevisiae. The extracts of yeast cells were purified by CsCl density gradient centrifugation after induction and cell lysis.

RESULTSElectrophoresis and sequencing analyses showed that the genes P1 and 3CD of poliovirus type I were successfully inserted into expression vector and encode a protein whose amino acid sequences were identical with wide-type genes of poliovirus type I. Electronic microscopy analysis showed that the VLPs of poliovirus type I could be efficiently formed in Saccharomy cescerevisiae.

CONCLUSIONThe VLPs of poliovirus type I could be efficiently produced by co-expression of P1 and 3CD genes in Saccharomy cescerevisiae.

Female ; Gene Expression ; Humans ; Male ; Poliomyelitis ; prevention & control ; virology ; Poliovirus ; genetics ; metabolism ; Poliovirus Vaccines ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virion ; genetics ; metabolism

Gout ; therapy ; Humans ; Integrative Medicine

Gastrointestinal neuroendocrine tumor(GI-NET)originates from peptide neurons and neu-roendocrine cells in gastrointestinal tract,and secrets peptide hormones,leading to carcinoid syndrome which rarely happens in clinical practice. Because of the improvement of diagnostic method and understanding of this rare disease,the morbidity is rising in recent years. The main treatments of GI-NET are surgery and compre-hensive therapy,consisting of chemotherapy and targeted therapy.

One of the important approaches for further prolonging remission duration and eradicating minimal residual disease in acute leukemia is immunotherapy. Four kinds of immunotherapy for acute leukemia are under investigation: (1) monoclonal antibodies, among them, Mylotarg (cytotoxic antibiotic calicheamicin linked to CD33 Mab) is given for the treatment of refractory or relapsed acute myeloid leukemia and molecular relapse in acute promyelocytic leukemia with good results, Campath-1H (antiCD52 Mab) is administered in the treatment of prolymphocytic leukemia and Rituximab (anti-CD20 Mab) in B-PLL with high complete remission rates. Other Mabs under preclinical and clinical trials include anti-IL-2 receptor Mab for the treatment of acute T lymphocytic leukemia, anti-220 kD Mab-6G7 for acute leukemias, recombinant immune toxin BL22 (anti-CD22) for hairy cell leukemia and Mabs labeled with radio-isotopes for different types of acute leukemias; (2) adoptive cellular immunotherapy using cytokine-induced killer cell, alloreactive NK cells, allogeneic or autologous leukemic-specific CD8(+) cytotoxic T lymphocytes, and other immune effector cells; (3) cytokines and other immune modulators comprising IL-2, IL-12, GM-CSF, CD40L, FLT-3L and thalidomide and its derivatives; (4) leukemia vaccines of several different formulations including antigen-specific, leukemia cell-based, leukemia antigen-pulsed dendritic cell (DC) and leukemia-derived DC vaccines, the latter two formulations are more attractive. In conclusion, up to now, the most effective example of immunotherapy in acute leukemia is provided by the administration of Mabs, and the majority of other approaches in immunotherapy for acute leukemia although promising, need further studies.
Acute Disease ; Adoptive Transfer ; methods ; Antibodies, Monoclonal ; immunology ; therapeutic use ; Cancer Vaccines ; therapeutic use ; Humans ; Immunotherapy ; methods ; trends ; Leukemia ; immunology ; therapy

Objective To study the reliability and validity of using sacral markers in evaluating the balance function in standing and walking of stroke patients with hemiplegia.Methods Twenty-one hemiplegic stroke patients were recruited and their baseline mean sway amplitude (MSA) and mean sway velocity (MSV) were measured using sacral markers and center of gravity analysis assuming a segmented body,thegold standard for such analysis.The data were analyzed using Bland-Altman plots to obtain the 95％ limits of agreement (LOA).Results ①Test-retest reliability:The 95％ LOA of the MSA in standing was (-4.42,5.14) on the X axis,(-6.04,4.52)on the Y axis,and (-1.75,1.31) on the Z axis.The MSV in standing was (-0.08,0.09) on the X axis,(-0.10,0.08) on the Y axis and (-0.03,0.02) on the Z axis.The 95％ LOA of the MSA in walking was (-185.74,105.53) on the X axis,(-22.57,2.76) on the Y axis and (4.43,2.76) on the Z axis.The MSV in walking was (-3.10,1.76) on the X axis,(-0.38,0.54) on the Y axis and (-0.07,0.02) on the Z axis.②Validity:The 95％ LOA of the MSA in standing was (-3.62,2.55) on the X axis,(-3.95,3.94) on the Y axis and (-7.35,19.43) on the Z axis.For the MSV in standing it was (-0.06,0.04) on the X axis,(-0.07,0.07) on the Y axis and (-0.12,0.32) on the Z axis.The 95％ LOA of the MSA in walking was (-4.40,4.74) on the X axis,(-17.35,4.14) on the Y axis and (-17.35,4.14) on theZ axis.For the MSV in walking itwas (-0.07,0.08) on the X axis,(-0.29,0.07) on the Y axis and (-0.12,0.18) on the Z axis.The 95％ LOAs of the variables representing their reliability and validity are small enough to be acceptable in clinical application.Conclusions The sacral marker method can be used in assessing the balance of stroke patients.

Objective To explore the extent of agreement between measurements of temporal and spatial gait parameters made with portable gait analysis equipment and in the laboratory.Methods Fifteen healthy young people submitted to laboratory gait analysis using 3D motion analysis apparatus and then on the same day to analysis using the Gait Watch portable apparatus.Cadence,stride length,walking speed and step length were recorded.Intraclass correlation coefficients (ICCs) and Bland-Altman plots were used to evaluate the agreement between the two gait analyses.Results Test-retest comparisons with the Gait Watch apparatus generated ICCs for the temporal and spatial parameters ranging between 0.80 and 0.98,indicating good test-retest reliability.Bland-Altman plots comparing the two measurement systems also showed good agreement.According to paired simple t tests,the stride length,walking speed,and step length assessments with the two systems showed significant differences.All exceeded the minimum detection threshold (stride length =0.05 m,walking speed =0.12 m/s,left step length =0.03 m,right step length =0.04 m).Conclusions Measurements of cadence,stride length,walking speed and step length with the two systems yield acceptable agreement,and either can be used in clinical walking assessment.