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Objective To construct the eukaryotic vector that expressing hepatitis B virus (HBV) S and the single fragment of variety chain (ScFv) of monoclonal antiboy against cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4) and to analyze the immunological activity of recombinant S-ScFv protein.Methods The oringially constructed pSect2/ScFv4F10 and pSect2/S were double enzyme digested by Sfi I and Hind Ⅲ,respectively.Then the HBV S gene was cloned into the pSect2/ScFv4F10 vector.The pSect2/ScFv4F10 and pSect2/S-ScFv4F10 were expressed in Chinese hamster ovary (CHO) cells,and the expressed proteins were verified through sodium dodecyl sulfatepolyacrylamide gelelectrophoresis(SDS-PAGE)andWesternblotting.Afterultrafiltration concentration and affinity chromatography,the biological affinity of the expressed ScFv4F10 and S-ScFv4F10 proteins were examined by competitive enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology.The comparison between groups was done by One-way ANOVA.ResultsThe eukaryotic expression vector of pSect2/S-ScFv4F10was successfully constructed,and relative molecular mass of the expressed protein of S-ScFv4 F10 was about 52 000 that analyzed by SDS-PAGE and Western blotting.With the fixed concentration of 4F10-mAb against CTLA-4,the A570 value of the mixed reaction with purified CTLA-4 antigen gradually increased with the decrease of ScFc fusion protein proportion; when the molar ratio of ScFv,S-ScFv4F10∶4F10=2∶1,the competitiveinhibitionratesagainst 4F10conjugatedantigenwere72.6％and64.5％,respectively.The affinity constants of association kinetics for CTLA-4 mAb,ScFv4F10 and S-ScFv4F10 with CTLA-4 antigen were 7.29 × 108 mol/L,9.52 × 106 mol/L and 2.04 × 106 mol/L,respectively,and the dissociation constants of KD were 1.40 × 10-9 mol/L,1.05 × 10-7 mol/L and 4.91 × 10-7 mol/L,respectively.ConclusionsThe eukaryotic expression vector of pSect2/S-ScFv4F10is successfully constructed,and the recombinant protein of S-ScFv4 F10 has a fairly high affinity with CTLA-4 antigen.

AIM:To optimize the I?B? mutant(I?B?M)gene derived from human placenta tissue by deleting N-terminal phosphorylation sites of serine 32/36,and to construct and identify its replication-deficient recombinant adenovirus(AdI?B?M).METHODS:The I?B?M gene(203-1 003 bp)was acquired by positional cloning,followed by subcloning it into pShuttle and pGEM-T vectors for further PCR,double digestion,DNA sequencing and homology analysis.Subsequently,the expression unit of pShuttle-I?B?M containing CMV promoter,I?B?M cDNA and poly A signals was inserted into Ad5 vector,after which the resultant recombinant adenovirus AdI?B?M was packaged in 293 cells by cotransfection with lipofectamine.Western blotting analysis and electrophoretic mobility shift assay were utilized to detect the AdI?B?M-mediated expression of I?B?M gene in 293 cells and its suppressive effect on phorbol myristate acetate(PMA)-induced nuclear factor ?B(NF-?B)activation in ECV304 cells,respectively.RESULTS:The relevant nucleotide and amino acid sequence of I?B?M gene was consistent with that of GenBank(accession number M69043).The titer of the prepared AdI?B?M was 4.0?10 12 pfu/L.Moreover,the I?B?M gene was expressed in 293 cells,and potently inhibited the PMA-induced NF-?B activation in ECV304 cells in a dose-dependent manner.CONCLUSION:AdI?B?M is a nonvel vector for both efficient transfer and expression of I?B?M gene as well as specific inhibition of NF-?B activity,providing a promising future for gene therapy of asthma.

Objective To evaluate the significance of combined detection of leptin (LEP),tumor necrosis factor-α (TNF-α),carcino-embryonic antigen (CEA),C-reactive protein (CRP) and interleukin-6 (IL-6) in both serum and pleural effusion in differential diagnosis of tuberculosis and malignant tumor.Methods LEP,TNF-α,CEA,CRP and IL-6 levels in both pleural fluid and serum samples from 95 cases of pleural effusion (including 54 cases of malignant pleural effusion and 41 cases of tuberculous pleural effusion) were detected by immunoturbidimetry and enzyme linked immunosorbent assay (ELISA),respectively.The data with normal distribution and skewed distribution were analyzed by t test and rank sum test,respectively.Results In patients with tuberculosis and malignant tumor,significant difference was observed in serum LEP [(0.42±0.47) ng/ mL vs (1.80±0.91) ng/mL,t=9.666,P=0.0001],TNF-α [(30.88±24.72) pg/mL vs (59.83±30.93) pg/mL,t=4.917,P=0.0001],CEA [(0.22±0.30) ng/mL vs (5.67±3.60) ng/mL,t=ll.074,P=0.0001] and IL-6 [(146.48±54.90) pg/mL vs (20.51±11.62) pg mL,t=-14.449,P 0.0001] levels.Serum CRP levels of patients with tuberculosis and malignant tumor were comparable [(32.78±22.43) mg/mL vs (37.80±16.74) mg/mL,t =1.249,P=0.215].In pleural effusion of the two groups (tuberculous pleural effusion vs malignant pleural effusion),LEP [(0.69±0.65) ng mL vs (4.33±2.16) ng mL,t 11.711,P 0.0001],TNFα [(20.60±17.80) pg/ mL vs (40.40±20.60) pg/mL,t=4.926,P=0.0001],CEA [(0.10±0.17) ng/mL vs (4.02±2.49) ng/ mL,t=11.537,P=0.0001] and IL-6 [(87.80±48.40) pg/mL vs (9.30±5.50) pg/mL,t =-10.333,P=0.0001] levels were significantly different,while CRP levels [(27.34±17.93) mg mL vs (29.60± 13.40) mg mL,t =0.709,P =0.102] were comparable.Conclusion LEP,TNF-α,and CEA levels in both pleural effusion and serum samples from patients with malignant tumor are higher than those with tuberculosis,while IL 6 levels are quite opposite.Combined detection of these parameters can help the differential diagnosis of tuberculous and malignant pleural effusion.

Objective To summarize the experience of improved nonmyeloablative hematopoietic stem cell transplantation(NSCT) in the treatment of severe aplastie anemia. Methods Seventeen patients with Severe Aplastic Anemia received NSCT after a nonmyeloablative conditioning.The patients were conditioned with decreased dosage of immunosuppressive agents of CTX and anti-lymphoid cell globulin or anti-thymus gland cell globulins;CsA and MMF were used to prevent the graft-versus-host disease (GVHD) after transplantation. Results 75 % of severe aplastic anemia in patients with type Ⅰ and 60 % of severe aplastic anemia in patients with type Ⅱ achieved rapid hematopoietic reconstitution, with good prognosis and implantation rate with fewer complieations and light symptoms were observed. Conclusion NSCT is an effective treatment of severe aplastic anemia.

Objective To investigate the efficiency and safety of intervention with flexible bronchoscope under general anesthesia by using laryngeal mask in patients with severe tracheal stenosis induced respiratory failure.Methods A total of 16 in-patients with respiratory failure caused by severe tracheal stenosis admitted from September 2009 to March 2012 were retrospectively reviewed.A comprehensive bronchoscopic intervention for the complete patency of airway was successfully performed with various techniques such as cryotherapy,electrocautery,balloon dilatation,and implantation of selfexpanding metal stents under genersl anesthesia by using laryngeal mask.The efficiency of comprehensive bronchoscopic intervention and dyspnea score were evaluated by chest CT scan and bronchoscopic examination before and after the treatment.Data were expressed as ((x-)± s).Paired t test was used for statistical analysis of the data.Results The degrees of tracheal stenosis and dyspnea score before and after intervention were (85.0±8.4)％ vs.(20.9±7.6)％ (P＜0.01) and (3.9±0.3vs.2.4±0.5,P＜ 0.01),respectively.There were no life-threatening complications occurred including massive haemorrhage.Conclusions It is an effective and safe technique to resolve the tracheal stenosis-induced respiratory failure with intervention by using flexible bronchoscope under general anesthesia with laryngeal mask,and it is a promising interventional treatment for clinic application.

Objective To express VP1recombinant protein of Echovirus 30 (ECHO30) in E.coli BL21(DE3) and to develop an enzyme-linked immunoassay (EIA) based on the recombinant antigen for detecting antibodies to ECHO30.Methods The target VP1gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR).The PCR products of the gene were inserted into pET44a vector,and then expressed in E.coli BL21( DE3 ).The purified recombinant protein was used for the development of EIA.Results The molecular weight of the recombinant protein was 95 000,and the antigenicity of which was identified by Western blot and EIA.Conclusion The recombinant protein VP1has been successfully expressed and purified,which can be used as diagnostic antigen.

Objective To study the function of core protein (CORE) of genotype 1b hepatitis C virus (HCV) of different strains (T: derived from tumor tissues; NT: derived from non-tumor tissues; C191: HCV-J6) and different domains (1-172, 1-126, 1-58, 59-126, 127-172 AA) of T CORE in the pathogenesis of HCV infection and to find the therapy target. Methods Different truncated genotype 1b HCV CORE eukaryotic expression plasmids (T, NT, C191) and different domains of T CORE were constructed and transfected to HepG2 cells. Cell apoptosis and necrosis were quantified by flow cytometry. Cell growth curves were observed with real time cell growth instrument. Results COREs from different strains of genotype 1b and different domains of CORE induced cell apoptosis and necrosis, and inhibited HepG2 cell growth at different levels. CORE derived from T induced apoptosis and necrosis and inhibited cell growth higher than that derived NT and C191. N terminal 1-58 AA of CORE derived from T induced cell apoptosis and necrosis and inhibited cell growth higher than any other domains. Conclusion COREs from different strains of genotype 1b HCV and different domains of CORE from the same HCV strain play different roles in their molecular pathogenesis of HCV. Among different domains of CORE, N terminal 1-58 AA might play an important role in its pathogenesis and be one target of gene therapy.

Objective To investigate the clinical features,diagnosis,treatment and prognosis of Dementia with Lewy Body (DLB) in order to improve doctors' understanding and experience.Methods Thirteen cases of clinicall diagnosed DLB were analyzed.The clinical manifestations,neurological examination,laboratory tests,treatment and prognosis were analyzed.Results All of 13 patients had fluctuating dementia.Among them,11 were diagnosed with probable DLB,and 2 were diagnosed with possible DLB.There were 8 cases (8/13) with volatility cognitive impairment,8 cases (8/13) with Parkinson's syndrome,8 casess (8/13) with visual hallucinations,13 cases (13/13) with memory loss,4 patients with limb tremor (4/13),5 cases with delirium (5/13),3 cases with increased sleep (3/ 13),1 case with silence (1/13),9 cases with anxiety and depression (9/13),one case with dizziness (1/13),6 cases with abnormal dysplasia (6/13),and one case with abnormal sensitive to diazepam (1/13).The cognitive function fluctuated,the condition deteriorated and two patients died during the follow-up from 6 months and 5 years.Conclusions Lewy body dementia is an irreversible and progressive neurodegenerative disease.Comprehensive understanding of its clinical features is helpful for the early diagnosis and treatment,which may in turn improve the prognosis.

Tuberculous meningitis (TBM) is one of the most serious and lethal diseases in neurology. The early diagnosis and treatment of TBM still faces severe challenges. Acid-fast staining of cerebrospinal fluid and culture of Mycobacteriumtuberculosis are the "gold standard" for the diagnosis of TBM, but lack sensitivity. With the application of metagenomic next-generation sequencing technology, significant advances have been made in the early diagnosis of TBM. The optimal treatment regimen for TBM remains to be further explored and studied. This article intends to provide an overview of the pathogenesis, pathology, clinical manifestations, diagnosis and treatment advancement of TBM, and provide guidance for clinical practice.