7.The construction, expression and immunological activity study of eukaryotic vector expressing cytotoxic T lymphocyte-associated antigen-4 ScFv and hepatitis B virus-S gene
Guoping PENG ; Dangjiu ZHAO ; Cheng ZHOU ; Linfu ZHOU ; Zhi CHEN
Chinese Journal of Infectious Diseases 2012;30(6):324-329
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.
8.Treatment and follow-up study of long QT syndrome with syncope in 11 children
Yefeng WANG ; Zhi CHEN ; Yunbin XIAO ; Zhenya YAO ; Cheng WANG
Chinese Journal of Applied Clinical Pediatrics 2016;31(13):993-996
Objective To assess the clinical features and treatment of children with long QT syndrome (LQTS)and syncope.Methods Eleven cases of children with LQTS and syncope between January 2009 and July 2014 in Hunan Children′s Hospital were retrospectively analyzed for clinical features,treatment and long term follow -up.Results There were 11 cases of children with LQTS aged 4.0 -14.5(9.16 ±2.71)years,8 male and 3 female, with syncope more than once.The range of QTc was 460 -521(483.72 ±22.90)ms.For 3 cases of acquired LQTS,1 case was parathyroid hypothyroidism causing hypocalcemia,1 case was myocarditis complicated with third degree atrio-ventricular block,and 1 case showed atrial flutter receiving amiodarone post congenital cardiac surgery.All patients re-covered after the inducement removed and primary illness cured.For 8 cases of congenital LQTS,3 cases of LQTS un-derwent genetic test (1 case of KCNQ1 gene mutation,2 cases of KCNH2 gene mutation).One case died after frequent torsade de pointes (Tdp)and ventricular fibrillation during hospitalization,the remaining 7 patients were given oral pro-pranolol,potassium chloride sustained -release tablets after discharge.Follow -up time was 8 to 75 months,an average of (45.73 ±24.42)months.One case died suddenly at home after 25 months of follow -up.The remaining 6 cases of children with congenital LQTS could withstand general activities without syncope,in which 4 cases had normal QTc by electrocardiography(ECG),and the findings in 2 cases did not change compared with those previously.The QTc re-turned to normal in children with acquired LQTS in the follow -up review.Conclusions Children with congenital LQTS should receive early genetic screening and genotyping for rational use of drugs.For children with higher risk of sudden death,drug therapy combined with implantable cardioverter defibrillator should be considered.For acquired LQTS,it should be better to remove the inducement and treat primary disease actively.
9.Analysis of Differentially Expressed Genes between Chronic Hepatitis B and Asymptomatic Hepatitis B Virus Carrying in Children
cui-lan, TANG ; zhou, ZHOU ; zhi, CHEN ; cheng-ji, LU
Journal of Applied Clinical Pediatrics 2006;0(16):-
Objective To explore the difference of pathogenesis between chronic hepatitis B (CHB) and asymptomatic hepatitis B virus(HBV) carriers(ASCs) in children.Methods Subtracted libraries was constructed by suppression subtractive hybridization (SSH) from peripheral blood mononuclear cell (PBMC) of two groups, beta-2-microglobulin (?_2-MG) was screened as one of differentially expressed genes. Serum was collected from children with CHB and ASCs, the differential expression of ?_2-MG was confirmed by enzyme immunoassay.Results Subtractive libraries were constructed successfully, the differentially expression of ?_2-MG and lactroferrin were up-regulated in CHB children.And the differentially expression of ?_2-MG on the level of protein was confirmed.Conclusion The up-regulation of ?_2-MG in CHB children is involved in the pathogenesis mechanisms.
10.Survey on and Phytogeny of Alicyclobacillus Species in Hot Springs of Southern China's Guangdong and Yunna Provinces
Zhi-Wei CHEN ; Cheng-Ying JIANG ; Shuang-Jiang LIU ;
Microbiology 1992;0(03):-
12 isolates of thermoacidophilies were obtained from samples of hot springs of Southern China's Guangdong and Yunna provinces. All isolates are heterotrophic. Cells are rods, Gram positive or variable. The optimal pH and temperature for growth are 3.5-5.5 and 43℃-52℃, respectively. Based on their morphological physiological properties and their 16S rDNA sequences, they were identified as members of Alicyclobacillus.