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Objective:To construct eukaryotic expression plasmids of pneumocystis carinii p55-v3 and p55-v0 antigenic genes and to identify their expression in COS-7 cells at mRNA level.Methods:Pneumocystis carinii total RNA was used as the template to amplify p55-v3 and p55-v0 antigenic gene by RT-PCR.The products were connected to pTA2 vector and then cloned in pVAX1 eukaryotic expression vector to construct recombinant plasmids as pVAX-p55-v3 and pVAX-p55-v0.After propagated in E.coli DH5α,the recombinant plasmids were transfected into COS-7 cells.After 24 h incubation,the RT-PCR was performed to identify the mRNA expression of p55-v3 and p55-v0 antigenic gene.Results:The recombinant plasmids were qualified by restrictive endonuclease digestion and sequencing.And when compared with that in GenBank,the homology of p55-v3 antigenic gene was 99.9% in nucleotides and 100% in amino acid.The homology between p55-v0 antigenic gene and the one reported previously in nucleotide and amino acid seguence were 99.8% and 100%.The results of RT-PCR confirmed that p55-v3 and p55-v0 antigenic genes were transfected into COS-7 cells successfully and the genes were expressed in the cells.Conclusion:In this study,the recombinant plasmids of pVAX-p55-v3 and pVAX-p55-v0 are conducted successfully and expressed in the COS-7 cells,which provide a basis for clarification of immunologic function of p55-v3 and study of DNA vaccine.
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This study sought to amplify the CDS region of p55-v0 and p55-v3 gene in Pneumocystis carinii (PC) and to analyze their sequences.The total RNA extracted from PC- infected lungs of rats were used as the template to amplify these genes by RT-PCR.The amplified products were connected to T-vector and transformed into E.coli DH5α.The recombinant T-vectors were selected in LB culture medium containing ampicillin.Then the positive clones of recombinants were identified by PCR and digested by restrictive endonuclease.After that the recombinant were sequenced and analyzed.It was found that the CDS region of p55-v0 gene contained 1245 bps,encoding 414 amino acids.The homology between this sequence and the one reported previously in nucleotide and amino acid were 99.8% and 100% respectively.Meanwhile,the CDS region of p55-v3 contained 1053 bps,encoding 350 amino acids.As compared with GenBank,the homology in nucleotide and amino acid was 99.9% and 100% respectively.However,the similarity in nucleotide between p55-v3 and p55-v0 was just 20.9%.In this study,the CDS regions of p55-v0 and p55-v3 gene were cloned successfully,and the high homology was found between p55-v0,p55-v3 and the one reported previously by sequence analysis,p55-v3 was different from p55-v0 in nucleotides and amino acids.This result would provide a basis for comparison of immunologic functions so as to explore the mechanism of action of the p55-v3 gene.
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Objective To Clone,express,purify 38 ku protein of mycobacterium tuberculosis,to study its immunological characteristics,and to evaluate its potenial value for serodiagnosis of tuberculosis.Methods Extract DNA from standard strain of H37Rv as the template,amplify gene of 38 ku protein by PCR,insert to PET-30a(+)and construct the recombinant plasmid,express 38 ku protein in E.coli BL21(DE3),purify by Nickel affinity chromatography,at last get target proteins of higher purity,analyze its immol/Lunological characteristics by Western blotting and ELISA technology from Feb.2003 to Mar.2004. Results The clone was analyzed at the nucleotide lever and showed the same DNA sequence coding for natural 38 ku protein. The recombinant protein expressed in inclusion body in E.coli BL21(DE3). The purity of terget protein was 92.7% by Nickel affinity chromatography,Western blotting assays showed that the recombinant protein had satisfactory antigenicity . 38 ku protein detected TB postive and negative refference serum based on the mechanism of indirect ELISA,results showed that the sensitivity was 80.5%(33/41)and the specificity reached to 96%(25/26).Conclusion The recombinant protein expressed in inclusion body in E.coli BL21(DE3)and had satisfactory antigenicity,and might be selected as one of serodiagnostic antigen of tuberculosis.
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0.05), but for the DCs with low DC-SIGN expression, the ability was much lower than those of the other group did (P
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Objective To investigate the inhibitive effects of biosynthesized short hairpin RNA(shRNA)on the duplication of Influenza A virus(IAV)in human bronchial epithelium(HBE)cells.Methods NP-shRNA and PA-shRNA targeting to the highly conservative sequences of IAV nucleoprotein(NP)and acidic RNA polymerase(PA)were designed and biosynthesized by in vitro transcription,and then were inserted into the plasmid pGenSil-1.After sequencing analysis,the recombinant plasmids NP-shRNA and/or PA-shRNA were transduced into HBE cells followed by infected with IAV A/PR/8/34 H1N1(A/PR8)virus.The cytopathogenic effect(CPE)of the HBE cells,hemagglutination assay(HA)and 50% tissue culture infective dose(TCID50)titer of A/PR8 in the culture supernatants were determined respectively.The mRNA and protein expressions of NP and PA were detected by RT-PCR and Western blotting respectively,and the results were compared in order to evaluate the inhibitive efficiency of NP-shRNA and/or PA-shRNA to A/PR8 duplication in HBE cells.Results The content of NP-shRNA and PA-shRNA biosynthesized by in vitro transcription were respectively 59.4 nmol and 50.6 nmol in each 100 ?l.The purity was more than 2.0 and the sequences were verified to be correct after sequencing analysis.The CPE of HBE cells and the virus titer in the culture supernatants of cells transfected with NP-shRNA,PA-shRNA or NP-shRNA+PA-shRNA were markedly lower than those of the control groups.In 3 h after 1 TCID50 A/PR8 infection,the mRNA level of NP in NP-shRNA group,the mRNA level of PA in PA-shRNA group and those in NP-shRNA+PA-shRNA group were 41.7%,43.4%,68.5% and 73.7% respectively,lower than those of the control group.At 48 h after 1 TCID50 A/PR8 infection,NP synthesis were 92.3%,84.0% and 91.7% respectively of those of the control group.Conclusion Anti-IAV NP-shRNA and PA-shRNA are successfully prepared by in vitro transcription.Both of those markedly inhibit the reproduction of IAV A/PR8 and produce a favorable protective effect on HBE cells.
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Objectives To study if there exists the ununi formity of drug resistance of mycobacterium tuberculosis (M.TB) isolated from MDR-TB patients .Methods One MDR-TB patient was chosen for this study a nd his sputum was cultured.The suspension of the culture was filtered through 8 0?m -pore-size membrane filter and single M.TB suspension was obtained.The single M.TB suspension was cultured on Lowsenstein Jensen (L-J) medium.24 of col onies were obtained and they were cultured respectively again to get pure strain s.The drug sensitivity test was carried out for these 24 pure strains of M.TB wi th Chinese standard method.H 37 Rv was taken as control.Results 1 strain (4 2%) was found to be susceptive to INH,22 strains ( 91 6%) resistant to low concentration INH (H 1 ),1 strain (4 2%) resistant to high concentration INH (H 10 ).2 strains (8 3%) were found t o be susceptive to RFP,2 strains (8 3%) resistant to low concentration RFP(R 50 ),20 strains (83 4%) resistant to high concentration RFP(R 250 ).We f ound 1 strain (4 2%) susceptive to SM,23 strains (95 8%) resistant to high concentr ation SM(S 100 ).All were susceptive to EMB.Conclusions The ununiformity of drug resistance exists among the M.TB isolated from the MDR-TB patients.Sensitive and resistant strains exist together,low and high concentration-drug-resistant strains coexi st in the same situation.The majority of M.TB is drug-resistant and only a few are still sensitive to low concentration- drug- resistant.
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Glucocorticoid (GC) has been widely used in treatment of respiratory diseases due to its high effectiveness. However, severe side effects can be caused if it is administered inappropriately. This paper reviews and discusses the effects and side effects as well as the clinical applications of GC.
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Objective To investigate the risk factors for nosocomial pulmonary fungal infection(PFI) in COPD patients.Methods The data of 44 cases of nosocomial PFI were analyzed from Jan 2000 to Jun 2003 in Department of Pulmonary Medicine,the First Hospital of Chongqing Medical University.44 cases of COPD patients were randomized as control.Results According to univariate analysis ,the risk factors associated with nosocomial pulmonary fungal infection include those of long-term use of broad-spectrum antibiotics,long-term use of adrenocortical steroid,diabetes mellitus,type Ⅱ respiratory failure,mechanical ventilation and hypoalbuminemia.But according to multivariate logistic regression analysis long-term use of broad-spectrum antibiotics,hypoalbuminemia,mechanical ventilation diabetes mellitus were risk factors.Conclusion Long-term use of broad-spectrum antibiotics,hypoalbuminemia,mechanical ventilation and diabetes mellitus are independent risk factors for nosocomial pulmonary fungal infection in COPD patients.
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AIM: To study the effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs. METHODS: 30 guinea pigs were divided into normal saline (NS) group, asthma group and M. vaccae treatment group at random, every group included 10 guinea pigs. Guinea pigs in M. vaccae treatment group were injected intramuscularly with 22 5 ?g M. vaccae 10 days before OVA immunization. TdT-mediated dUTP nick end labeling (TUNEL) technique was used to investigate the apoptosis of eosinophils and immunohistochemistry method was used to study the expression of Bcl-2 protein in lung tissues. RESULTS: The apoptosis index (AI) of eosinophils in lung tissues in M. vaccae treatment group was significant higher than that in asthma group (23 78?5 42)% vs (4 56?0 68)%, P
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Aim To study the immunoregulatory effect of Mycobacterium vaccae vaccine on asthmatic guinea pig.Methods 30 guinea pigs were randomly divided into NS,asthma and Mycobacterium vaccae vaccine groups. A guinea pig model of asthma was formed with ovalbumin.Every guinea pig of Mycobacterium vaccae vaccine group was intramuscularly injected with 22.5 ?g Mycobacterium vaccae vaccine 10 days before ovalbumin immunization.mRNA expressions of IL-4,IL-5 and IFN-? in lung tissue,total IgE and ovalbumin-specific IgE in serum were determined. Results For Mycobacterium vaccae vaccine group guinea pigs, mRNA expressions(optical density value) of IL-4(0.060?0.018) and IL-5(0.052?0.006)in lung tissue were significantly lower than those of asthma group(0.111?0.025,0.106?0.030 respectively; both P
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Objective:To construct the eukaryotic vector of human DC-SIGN and EGFP fusion protein,and to identify the protein in cell line COS7.Methods:RT-PCR,T-vector and pEGFP-C1 vector were used to construct the recombinant expressing plasmid encoding for the fusion protein of DC-SIGN and EGFP.COS7 cells were transfected with the plasmid.Real-time PCR and laser scanning confocal microscope were used to quantificate expression of the fusion protein and cell function of uptaking BCG.Results:DC-SIGN cDNA was successfully cloned into the eukaryotic vector pEGFP-C1.The recombinant vector was transfected into COS7,real-time PCR test showed the amount of mRNA encoding for the fusion protein was 4.52?1011 copies/ml and laser scanning confocal microscope confirmed that the cells could uptake BCG.Conclusion:We have constructed a recombinant vector expressing DC-SIGN and EGFP fusion protein.