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Objective To establish a dual real-time fluorescence quantitative polymerase chain reaction (dual real-time PCR) assay to detect human vitamin D receptor (VDR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Methods GAPDH gene was used as the internal control. The specific primers and TaqMan probes were designed by Primer Premier 5.0 software, which were applied to detect the VDR/GAPDH mRNA levels. The obtained PCR products were puri-fied to construct the VDR/GAPDH recombinant plasmid, which was taken as the standard to analyze the sensitivity and re-peatability of the method. Results The amplification products were confirmed as the specific fragment of VDR/GAPDH by DNA sequencing instrument. The results showed that the sensitivity, linear range, the determinate coefficient, the amplifica-tion efficiency, the intra-assay and inter-assay coefficient of variation were 40 copies/μL, 4.00 × 101-4.00 × 105 copies/μL, 0.998, 96.10%, 0.09%-1.21%, 0.17%-0.51%for VDR, and 40 copies/μL, 4.00 × 101-4.00 × 105 copies/μL, 0.999, 85.15%, 0.35%-0.88%, 0.51%-2.46% for GAPDH, respectively. Conclusion These results demonstrate that the dual real-time PCR assay with high sensitivity and specificity can detect the relative expressions of human VDR by single reaction tube, which can effectively shorten the time and reduce the experimental error.
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ObjectiveTo construct the eukaryotic expression plasmids containing cysteine protease inhibitor (CPI) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene from periodic Brugia malayi (Bm),and to observe its cellular immune response in mouse.Methods pcDNA3.1 (+)-BmCPI/BmGAPDH was constructed.The recombinant plasmids were screened and identified by digestion with restriction enzyme.BALB/c mice were injected intramuscularly with a dosage of 100 μg purified recombinant plasmid DNA with GpG oligodeoxynucleotide (CpG ODN) and two same doses were administrated at 2-week intervals.pcDNA3.1 (+) and phosphate buffered solution (PBS) were used as controls.The tissue of muscles at 4 weeks after the third injection was collected and the target gene was detected by reverse transcription-polymerase chain reaction (RTPCR).Two weeks after the third immunization,the stimulation index (SI) of spleen lymphocytes of immunized mice was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method and the serum levels of interleukin (IL)-4 and interferon (IFN)-γ were detected by enzyme-linked immunosorbent assay (ELISA).The data were analyzed by t test.ResultsBmCPI/BmGAPDH gene in the injected muscle of the immunized mice was detected by RT-PCR. At 6 weeks after immunization,the SIot spleen T lymphocytes in pcDNA3.1 (+)-BmCPI/CpG group and pcDNA3.1 (+)-BmCPI/BmGAPDH/CpG group were 1.466 ± 0.635 and 1.610 ± 0.112,respectively,which were both higher than PBS group and pcDNA3.1( +)-CpG group (1.004 ± 0.019 and 1.078 ± 0.129,respectively) (t=64.438,45.318,42.749 and 34.314,respectively; all P＜0.05).At 4 weeks after immunization,the serum levels of IL-4 and IFN-γ of mice in pcDNA3.1 ( + )-BmCPI/BmGAPDH/ CpG group were significantly higher than those in pcDNA3.1 (+)-CpG group (t=288.053 and 76.453,respectively; both P＜0.05),while the serum level of IFN-γ was also higher than that in pcDNA3.1 (+)-BmCPI/CpG group (t=129.642,P＜0.05). ConclusionThe recombinant eukaryotic plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH could be expressed in mice,and could elicit specific cellular immune responses in immunized mice.
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Objective To study whether G3PD modifier protein (MP) could repair tubular epidermal cell(TEC) and ameliorate renal functions in aged rats with gentamycin induced acute tubular necrosis(ATN). Methods Aged and young rats were randomly divided into 3 groups, namely aged and young normal groups, aged and young model groups and the aged and young MP groups. The animal models of acute renal failure (ARF) were induced by gentamycin (140 mg?kg -1 ?d -1 , ip?7 d). Nitric oxide (NO) concentration was determined by Cd activated cadmium reduction method, malonaldehyde(MDA) with thiobarbituric acid test, superoxide dismutase(SOD) with hydroxylamine test, serum creatinine (Scr) with picric acid method, and the renal histology was observed by light and electron microscopy. Results NO was significantly higher in the aged MP group than that in the aged model group 〔serum: (94.29?7.68)?mol/L vs (70.14?5.53)?mol/L, P
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AIM: To observe the levels of triglyceridemia(TG),uricemia(UA),glycemia(GLU),the activity of 3-glyceraldehyde phosphate dehydrogenase(GAPDH) in blood and the gene expression in the liver in the animal model of hypertriglyceridemia,hyperuricemia and hyperglycemia.METHODS: SD rats were randomly divided into three groups,control group,fructose group,fructose and fenofibrate treated group.Rats in control group were fed with standard chow.Rats in fructose group were fed with high fructose diet.Rats in fructose and fenofibrate group were fed with high fructose diet,and treated with fenofibrate 100 mg?kg-1?d-1 by intragastric administration at the same time.Rats in control group and fructose group were given distilled water by intragastric administration.The levels of TG,UA and GLU were detected.Improved method was used to measure the activity of GAPDH.Quanti Gene technology was applied to determine the transcriptional level of GAPDH mRNA.RESULTS: During 7-28 d,the level of TG in fructose group was significantly and persistently high.During 14-28 d,the level of UA was higher.The level of GLU higher than that in control group was only observed at 28th day.The GAPDH activity change in blood and the expression in liver were significantly lower than that in norma1 during 7-28 d.Fenofibrate had the effect on reducing TG only at 7th day and reduced the level of GLU significantly at 28th day.Fenofibrate also increased the GAPDH activity in blood and the expression in liver at 7th day.CONCLUSION: ① The level of TG is significantly and persistently high in the early days by feeding with excessive fructose.The levels of UA and GLU are higher with the time cause of the model development.② The significantly higher level of TG,UA and GLU may be correlated with the reduction of the GAPDH activity in blood and the expression in liver.③ Fenofibrate has the effect of reducing the TG level only in the condition of hypertriglyceridemia,but not in the condition of accompanying hyperuricemia and hyperglycemia.④ The mechanism of reducing the TG level by fenofibrate may be correlated with the increase in GAPDH activity in blood and the expression in liver.