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Objective By prokaryotic expression and purifying the human bocavirus recombinant protein VP2, to establish the indirect enzyme-linked immunoassay for detection of virus. Methods We amplified the human bocavirus recombinant protein VP2 gene fragments from WHL-1 template by PCR , and cloned into the expression vector pET28a, then conversed into the BL21 (DE3) and expressed the fusion protein detected by Western Blot detection , the obtained the antibody and detected the human bocavirus in serum in Guanghzhou area in healthy people. Results The Recombinant prokaryotic expression identified correct by double enzyme, and it could occur specific reaction with the virus positive serum. The best optimal antigen coating concentration were serum multiples and blocking BSA was 2 mg/mL , 1 ∶ 200 and 1%. The best working dilution of enzyme-labeled secondary antibody was 1 ∶ 4 000. The best working hours was 1h. This detection method had good specificity and reproducibility. The cut-off of the indirect ELISA method was 0.1 and the sensitivity and specificity of the developed ELISA method were 92% and 98% respectively. The coincidence rate of determination results by the developed kit and control kit was 97%. Conclusion The competitive ELISA established by prokaryotic expressing and purifying the human bocavirus protein VP2 protein , provides a basis in detecting the human bocavirus serum antibody.
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Objective To discuss the clinical value of the expression level of acute lower respiratory tract Boka virus (HBoV)in‐fectecd children whose detection serum specific antibody .Methods 904 cases of children with acute lower respiratory tract Boka vi‐rus infection hospitalized from March 2011 to July 2014 who were selected as study objects ,serum ,sputum ,bronchoalveolar lavage fluid HBoV DNA positive were as the gold standard for diagnosis of acute lower respiratory tract infection in HBoV ,the positive serum HBoV antibody of HBoV in children with acute lower respiratory tract infection was defined as the observation group ,serum HBoV antibody negative acute lower respiratory tract infection in children with HBoV was defined as the control group ,the correla‐tion between serum HBoV antibody and acute lower respiratory tract HBoV infection children whose clinical characteristics were analyzed .Results Serum HBoV antibody in the diagnosis of acute lower respiratory tract infection of HBoV whose sensitivity ,spe‐cificity ,positive predictive value ,and negative predictive value ,accuracy of diagnosis were separately 60 .32% 、90 .25% 、31 .67% 、96 .81% 、88 .16% .In the general data ,between the observation group and the control group in gender ,age ,hospitalization time , there were no significant differences(P>0 .05) .In the clinical manifestations ,nasal congestion and runny nose ,cough ,fever ,vomi‐ting and diarrhea ,shortness of breath ,breathing difficulties whose occur rates had no significant differences between the observation group and the control group(P>0 .05) ,the incidence of wheezing of the observation group was significantly higher than that of the control group ,the difference had statistical significance (P0 .05) .Conclusion Serum HBoV antibody is in favor of acute lower respiratory tract infection of HBoV in the diagnosis of exclusion ,and the serum HBoV antibody positive and acute lower respiratory tract infection of HBoV have a certain relationship in children with wheezing symptoms .
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Objective To discuss the correlation between the level of serum cystatin C and lipids in patients with system lupus erythematosus.Methods Used automatic biochemical analyzer to detect serum cystatin C (CysC),triglyceride (TG),total cholesterol (TC),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol (LDL-C)and hsCRP levels in 136 cases of SLE patients and 113 cases of healthy people.Data obtained using SPSS13.0 software to carry on sta-tistical analysis.Results Outcome of SLE patients group compared with healthy controls,hsCRP (13.5 ± 4.85 mg/L vs 2.03±0.88 mg/L),CysC (2.63±1.95 mg/L vs 0.85±0.37 mg/L),LDL-C (3.06±1.21 mmol/L vs 2.33±0.41 mmol/L),TC (5.32±2.63 mmol/L vs 4.02±1.67 mmol/L)and TG (1.92±0.83 mmol/L vs 1.44±0.8 mmol/L)were signifi-cantly higher the difference between groups was statistically significant(t=2.45~12.4,P <0.05).Compared with healthy controls,HDL-C (1.12±0.31 mmol/L vs 1.52±0.85 mmol/L)was decreased (P <0.01).In SLE patients group,the ser-um CysC level and hsCRP,TC,TG and LDL-C were positively correlated,and the level of HDL-C was negative to the level of CysC.The health control group was no significant correlation.Conclusion Serum lipid levels of SLE patients were posi-tive to the level of CysC.Suggest that joint detection of SLE patients serum CysC and blood lipids index is helpful to the di-agnosis of SLE treatment and condition monitoring.
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Objective To investigate clinical value of the anti-nuclear antibody (ANA ) ,anti-double strand DNA (dsDNA ) anti-body and anti-extractable nuclear antigen(ENA) antibody repertoire in systemic lupus erythematosus (SLE) diagnosis .Methods 158 SLE patients were served as SLE group and another 50 healthy people as the control group .Indirect immunofluorescence(IIF) and immunoblotting test(IBT ) were employed to detect ANA ,anti-dsDNA antibody and anti-ENA antibody repertoire .Results Positive rates of ANA ,anti-dsDNA antibody and anti-ENA antibody repertoire in SLE diagnosis were 81 .65% ,68 .35% and 87 .34% ,respectively ,which were all lower than that of their combined detection (93 .04% ) ,with statistically significant difference (P<0 .05 ) .Among anti-ENA antibody repertoire ,the positive rate of anti-U1-nuclear ribonucleoprotein (U1-Nrnp ) antibody (52 .53% ) was the highest .Conclusion Combined detection of ANA ,anti-dsDNA antibody and anti-ENA antibodies repertoire has some clinical value of early diagnosis of SLE .
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Objective To evaluate the value of HbA2 level determined by capillary electrophoresis (Hb-CE)in screening and diagnosis of thalassemia.Methods HbA2 level of 249 thalassaemia carriers and 142 healthy controls confirmed by molecular biological detection were determined by Hb-CE method.The thalassaemia carrier subjects were divided into different groups and subgroups according to their results of gene detection.The sensitivity,specificity,accuracy,positive predictive value and negative predictive value for the diagnosis ofα-thalassemia,β-thalassaemia,α,β-thalassaemia were calculated under different HbA2 cut-off value.Results Mean value of HbA2 in healthy controls was (3.03±0.27)%.Mean values of HbA2 inα-thalassemia group and its subgroups of silentα-thalassemia,standardα-thalassemia and hemoglobin H disease were (2.38± 0.55)%,(2.61±0.46)%,(2.47 ± 0.32)% and (1.07 ± 0.17)%,respectively.Mean values of HbA2 inβ-thalassaemia group and itsβ0 subgroup,β+ subgroup were (5.65±0.47)%,(5.71±0.48)% and (5.56±0.43)%.Mean value of HbA2 in compoundαandβ-thalassaemia group was (5.7±0.82)%.Compared with healthy controls,HbA2 level inα-thalassemia group,silentα-thalassemia subgroup,standardα-thalassemia subgroup and hemoglobin H disease group decreased signifi-cantly (t values of 11.73,5.02,12.91 and 33.46,respectively,P0.05).HbA2 level inβ-thalassaemia group,β0 subgroup,β+ subgroup and compoundαandβ-thalassaemia group increased significantly (t values of 55.12,44.33,38.94 and 9.10,respectively,P0.05).Of 249 thalassemia carriers,all 124β-thalassaemia carriers were distinguished with ele-vated HbA2 level (>3.5%)determined by Hb-CE and only 57 were distinguished from 117α-thalassemia carriers by Hb-CE.Under the cut-off value of 2.5%,the sensitivity,specificity,positive predictive value,negative predictive value and accu-racy for the diagnosis ofα-thalassemia were 48.72%,97.18%,93.44%,69.70%,75.29%,respectively.Under the cut-off value of 3.5%,they were 100.00%,98.59%,98.41%,100%,and 99.25% for the diagnosis ofβ-thalassaemia,respectively. The analysis of ROC curve showed that the optimal HbA2 cut-off values for diagnosis ofα,β-thalassaemia by capillary elec-trophoresis were 2.8% and 3.7%,respectively.Conclusion When no abnormal bands,the elevated HbA2 (>3.7% in this study)determined by Hb-CE could be used as a marker forβ-thalassaemia diagnosis,but theβ-thalassaemia co-existingα-thalassemia could not be differentiated fromβ-thalassaemia diagnosis.Decreased HbA2 level (<1.5% in this study)and HbH band could be used for the diagnosis of hemoglobin H disease.Only HbA2 determination by Hb-CE has no clinical sig-nificance for the screen and diagnosis ofα-thalassemia.
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Objective To express the capsid proteins of WU polyomavirus(WUPyV) for research and find antigen for diagnostic value. Methods Coding sequences of capsid proteins of WU polyomavirus by PCR were cloned in prokaryotic expression vector PGEX-20T. Recombinant plasmids were transformed into E. coli BL21(DE3) and induced by IPTG for proteins expression. Recombinant proteins were identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed three bands with molecular relative mass of 69×103, 63×103 and 56×103. The recombinant proteins were recognized by anti-GST McAb. The antigenicity was tested by Western blot using 16 WU polyomavirus positive and 70 negative sera. Conclusion Recombinant VP1, VP2 and VP3 expressed in E. coli can combine with WUPyV-Ab and have good antigenicity. They can be used for further research.
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Objective To detect human metapneumovirus (hMPV)in respiratory intection rapidly and perform molecular analysis of hMPV.Methods Seven respiratory tract virus(11 subtypes)were assessed using multiplex PCR technology and flexible Multi-Analyte Profiling(suspension array).Human metapneumovirus was confirmed by using a real.Time reverse ranscriptase CR(RT-PCR)assay followed by sequencing.The cladogram analysis was performed further.Results The virus were detected in 40.2%(19/47)samples collected from clinicsl respiratory tract infections,including 8(42.1%)HRSV,7(36.8%)influenza virus,1(5.3%)parainfluenza virus,1(5.3%)rhinovirus,1(5.3%) coxsackievirus and 1(5.3%)human etapneumovirus infections.This is the first time that hMPV was deteced from clinical samples in Shenzhen.The sequencing of specific fragment of neucleoprotein of hMPV showed this hMPV shares over 98% homology with Beijing strain.Japan strain and Thailand strain.The cladogram analysis showed that they were in the same cluste.Conclusions Human etapneumovirus is a maior cause of children respiratory tract disease. Multiplex PCR technology and nexible Multi-Analyte Profiling were hish sensitive and high-throughput for detection of human metapneumovirus.They axe very robust and applicable in etiology analysis.
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Objective To analyze the antigens of solid cultured germ, liquid cultured germ and secreted composition, looking for the main immunity antigens of Mycobacterium tuberculosis H37Rv.MethodsShowing the difference of the antigens of solid cultured germ, liquid cultured germ and secreted composition by SDS-PAGE, Western blotting, monoclonal and polyclonal antibody techniques.Results Antigens of the solid cultured germ were nearly accordance with the liquid cultured germ and they were difference with secreted proteins. The main immunity antigens of the cultured germ were 79 000, 54 000 ,38 000~50 000, 28 000 and the secreted proteins are 66 000, 55 000~64 000, 30 000~34 000, 24 000, 16 000.The antigens of 66 000, 55 000 and 16 000 were proved that they were secreted proteins by monoclonal antibodies.Conclusions This study demonstrated that the main immunity antigens of the cultured germ in Mycobacterium tuberculosis H37Rv were difference with the secreted one .This is beneficial to the cognition of H37Rv and looking for special antigens for diagnosis of tuberculosis .
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AIM To observe the effect of intravenous injection of erythromycin (EM) on interdigestive migrating motor complex (MMC) and postprandial gastrointestinal contraction in conscious dogs , and to study its possible mechanism. METHODS Gastrointestinal contractile activity was recorded using low compliance capillary water perfusion manometric system. EM was administered intravenously during phaseⅠ and after meal, and blood samples were drawn for measuring plasma motilin concentrations. RESULTS ①Plasma motilin levels showed cyclical fluctuations in different phases of MMC, and plasma motilin reached peak during phaseⅢ and lowest during phaseⅠ.②EM induced phaseⅢ-like contractions in the antrum and duodenum, which was not accompanied by a peak in plasma motilin level. The optimum dose of EM for resulting in a premature phaseⅢ with the same characteristics as the spontaneously occurring phaseⅢ was established to be 0.5 mg*kg-1. The dose of 10 mg*kg-1 EM induced gastrointestinal continuous contractions and duodeno-gastric retrograde peristalsis which was associated with retching and vomiting. ③Atropine obviously inhibited EM-induced phaseⅢ activity in the antrum and duodenum. ④EM powerfully enhanced postprandial gastrointestinal contractile activity. CONCLUSIONS The results suggests that EM is a potent prokinetic agent. The mechanism is not related to the release of motilin, but may be mediated partially by cholinergic pathway.
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OBJECTIVE To explore the phenotype and genotype of Klebsiella and Escherichia coli producing extended-spectrum ?-lactamases(ESBLs).METHODS The producing ESBLs strains in 110 E.coli and 94 Klebsiella isolates were determined according to disk diffusion test,recommended by NCCLS and the drug-resistant genes such as TEM,SHV,CTX-M-1,CTX-M-2 and CTX-M-9 were detected by PCR and sequencing.RESULTS The producing ESBLs rates were 36.2%,42.7% and 39.7% in Klebsiella,E.coli,and in both two strains,respectively.The positive rate in detecting ESBLs by CTX and CTX/CLAV was higher(66.7%);the negative rates were 68.1% in E.coli,64.7% in Klebsiella when using CAZ alone and CAZ/CLAV.The rates of TEM,SHV,CTX-M-1,CTX-M-2 and CTX-M-9 were 54.3%,38.3%,21.0%,24.7% and 70.4%,respectively,in producing ESBLs isolations.The CTX-M genotype was predominant,(91.4%);the isolations(71.6%) contained more than two resistance genes.CONCLUSIONS More attention should be payed on ESBLs producing E.coli and K.pneumoniae strains.
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AIM To observe the effect of intra- venous injection of erythromycin (EM) on interdigestive migrating motor complex (MMC) and postprandial gastrointestinal contraction in conscious dogs, and to study its possible mechanism. METHODS Gastrointestinal contractile activity was recorded using low compliance capillary water per fusion manometric system. EM was administered intravenously during phase I and after meal, and blood samples were drawn for measuring plasma motilin concentra- tions. RESULTS ①Plasma motilin levels showed cyclical fluctuations in different phases of MMC, and plasma motilin reached peak during phaseⅢ and lowest during phase Ｉ. ②EM induced phase Ⅲ -like contractions in the antrum and duodenum, which was not accompanied by a peak in plasma motilin level. The optimum dose of EM for resulting in a premature phaseⅢ with the same characteristics as the spontaneously occurring phaseⅢ was established to be 0. 5 mg.kg-1. The dose of 10 mg.kg-1 EM induced gas- trointestinal continuous contractions and duodeno-gas-tric retrograde peristalsis which was associated with retching and vomiting. ③Atropine obviously inhibited EM-induced phase Ⅲ activity in the antrum and duodenum. GEM powerfully enhanced postprandial gastrointestinal contractile activity. CONCLUSIONS The results suggests that EM is a potent prokinetic agent. The mechanism is not related to the release of motilin, but may be mediated partially by cholinergic pathway.