ABSTRACT
ObjectiveTo prepare antiserum specific to aminoacy1-3″-adenylyltransferase [ AAD (3″) ],and to explore the application value of the prepared antiserum in detecting the expression levels of aadA2 gene that downstream of 8 different promoters (PcS,PcH2,PcH1,PcW,PcS-P2,PcH2-P2,PcH1P2 and PcW-P2 ) of variable regions in class 1 integron.MethodsaadA2 gene was amplified by polymerase chain reaction(PCR) and cloned into the expression plasmid pET19b.After inducing,the recombined aminoacy1-3″-adenylyltransferase[ AAD(3″)] with His-tag was expressed,purified and immunized rabbits to get anti- AAD(3″) specific serum.The prepared antiserum was used to detect the translation levels of aadA2 gene that downstream of different promoters of variable regions in class 1 integron by Western blotting (WB).Broth microdilution method was used to detect the minimal inhibitory concentrations (MIC) to streptomycin in Escherichia coli JM109 with aadA2 gene downstream of different promoters of variable regions.ResultsRecombined AAD (3″) expression plasmid pET19b-aadA2 was constructed successfully and was verified by sequence analysis.After transformed into E.coli BL21 ( DE3 ),a resoluble recombined AAD(3″) high expression strain was obtained.After fermentation,recombined AAD(3″) was purified and immunized rabbits.The anti- AAD(3″) specific serum was obtained with titer ＞ 1∶100 000.WB was used to detect the expression levels of AAD (3″),the translation product of aadA2 gene,that downstream of 8 different promoters of variable regions.The relative expression level of AAD (3″) that downstream of PcW was assumed to be 1,then the relative expression levels of AAD(3″),which all were detected 3 times independently,that downstream of PcS,PcH2,PcH1,PcS-P2,PcH2-P2,PcH1-P2 and PcW-P2 were 12.9±2.3,9.1±1.0,2.0±0.4,16.0±1.3,14.1 ±1.3,10.5±0.7 and 8.9 ±1.7 respective.Very different expression levels of AAD (3″) that downstream of different promoters of variable regions were obtained( F =32.421,P ＜ 0.01 ).The mean values of MIC,which all were detected 3 times independently,to streptomycin in E.coli JM109 with aadA2 gene downstream of PcS,PcH2,PcH1,PcW,PcS-P2,PcH2P2,PcH1-P2 and PcW-P2 were 256,256,64,128,32,128,4 and 64 mg/L respective.These results indicated the different expression levels of aadA2 gene that downstream of different promoters of variable regions can confer their host bacteria different resistance levels to streptomycin.Conclusions Resoluble recombined AAD(3″) is purified successfully and high titer anti- AAD(3″) specific antiserum is obtained from the immunized rabbits.This laid foundation for further investigation on the correlationship between the expressions of intI1 gene and the gene cassettes within variable regions.The expression levels of antibiotic gene cassettes that downstream of different promoters of variable regions are very different,so are the very different antibiotic resistance levels of the host bacteria.Therefore more attentions should be paid to the researches on the classification of promoters of variable regions when molecular epidemiology studies on the class 1 integrons in clinical isolates were conducted.
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Objective To investigate p16 promoter methylation in premature rats with chronic lung disease induced by hyperoxia. Methods Eighty premature Wistar rats were randomly divided into two groups: hyperoxia group (fraction of inspiratory oxygen) 0. 90 and control group (fraction of inspiratory oxygen 0. 21), 40 rats for each group. Semi-nested methylation specific polymerase chain reaction and methylation specific polymerase chain reaction were applied respectively to detect p16 promoter methylation in lung tissues. Additionally, p16 mRNA and protein expressions in lung tissue were detected by reverse transcription- polymerase chain reaction, Western blot and immunohistochemistry method. Results The methylation was not found in control group by seminested methylation specific polymerase chain reaction and methylation specific polymerase chain reaction, while was found in different aged rats of the hyperoxia group. The methylation detection rate was higher by using the semi-nested methylation-specific polymerase chain reaction (52.5%, 21/40) than that by methylation specific polymerase chain reaction (42.5%, 17/40) in the hyperoxia group,but there was no statistically significant difference between the two methods. The p16 mRNA in the hyperoxia group were significantly lower than in the control group at day 7, 14 and 21(1.73 ± 0.40 vs 2.11±0. 37,1.29±0. 19 vs 1.60±0. 27,0. 95±0.25 vs 1.72±0. 34, t=2.19, 2.95 and 10. 43,P＜0. 05). The p16 protein expressions by western blot in the hyperoxia group were significantly lower than in the control group at day 7, 14 and 21 also (88. 1±8. 7 vs 95.0±4.1,65.7±4.5 vs 83. 5±13.6 and 50.4±4.9 vs 86.7±11.9, t=2.27,3.95 and 13.40,P＜0.05). The expression of p16 mRNA (1.06±0.61) and protein (62.32±25.65) in lung tissues of rats with methylation was lower than that without methylation (1.63±0.62 and 94.93±22.21, respectively) (t=2.95, OR=0. 86;t=4.28, OR=0. 85,P＜0.01, respectively). Conclusions Exposure to hyperoxia might induce p16 promoter methylation in lung tissues in premature rats. Methylation risk increases as exposure time extends. p16 promoter methylation induced by hyperoxia might participate in the mechanism of lowering p16 mRNA and protein expression, but might not result in p16 gene silence.
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Objective To study anti-tumor effects on human ovarian cancer xenografted tumors in mice by constructing adenoviral expression vector containing canstatin gene and hTERT gene core promoter (AdhTERT-Can). Methods AdhTERT-Can vector was constructed and identified by means of enzyme cutting, electrophoresis and sequencing. Then, transfected into HO8910PM cells by means of lipofectamine and confirmed by green fluorescence protein(GFP) expression under laser confocus microscope. The mRNA expression of canstatin gene was tested by RT-PCR. Human ovarian cancer xenografted tumor models in nude mice were established and randomly divided into AdhTERT-Can group received viral supematant solution of AdhTERT-Can by tail vein injection, Ad-Can groups received viral supernatant solution of Ad-Can by tail vein injection, and control groups received phosphate buffer solution (PBS). The volume of tumors were measured and compared in each group to evaluated the anti-tumor efficacy. Results All the constructed vectors of AdhTERT-Can were verified by enzymed digestion. There were green fluorescence from 60% of HO8910PM cells transfected by AdhTERT-Can under laser confocus microscope, and the mRNA expression of canstafin gene in HO8910PM cells were also verified by RT-PCR The growth of tumor in AdhTERT-Can group was significantly inhibited compared with those in Ad-Can groups and control groups from the 8th day (P <0.01 ). However, there were not significant difference between Ad-Can group and PBS group (P> 0.05). On the 30th day, the tumors showed liquefaction necrosis and cystic degeneration in each group, especially in AdhTERT-Can group. Conclusions The recombinant adenovirus vector of AdhTERT-Can has been constructed successfully and could steady express in ovarian cancer cell lines HO8910PM. The results shown that it could inhibit significantly the growth of human ovarian cancer xenografted tumors in mice and shown to be the target of gene therapy.
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Objective To develop a clinically useful assay for detecting the mutations of HBV pre-C/BCP based on the pyrosequencing and accuracy, reproducibility and reliability of this assay was evaluated. Methods The pyrosequencing primers for HBV pre-C/BCP mutation were designed through the cluster analysis among one hundred HBV gene sequences. After the amplification of the fragment of pre-C/BCP with the template of pre-C/BCP mutation plasmids, the pyrosequencing method for pre-C/BCP detection was initially set up with this standard sample. The accuracy, reliability and reproducibility of the pre-C/BCP pyrosequencing were confirmed through the pre-C/BCP plasmids as a standard sample when compared with Sanger/genechip sequencing method pre-C/BCP pyrosequencing assay was applied for detecting pre-C/BCP mutation types of 60 chnical serum samples in HBV patients. Results The pre-C/BCP mutation detection assay based on pyrosequencing has been established in our study. The coincidence rate between pyrosequencing and Sanger squencing was 100%. The coincidence rate between the result of pyrosequencing and of genechip method was 91.7%. The reproducibility of this assay was 97. 8%. It indicates the pre-C/BCP pyrosequencing is a high-accurate method with, good-reproducibility and high-reliability. And multi-site detection can be achieved by pyrosequencing one time. A rare mutation T1758C was also detected. Conclusion Pyrosequencing for pre-C/BCP mutations assay is high-throughout method for simultaneous detection of multi-site mutation.