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Objective:To construct a eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi cysteine protease inhibitor/glyceraldehyde 3-phosphate dehydrogenase (CPI502/GAPDH720) multi-epitope gene and observe its protein expression in Hela cells. Methods:Primers were designed according to the predicted gene sequences of T cell epitopes. The target gene fragment was amplified by reverse transcription (RT)-PCR using plasmid pGEM-T-CPI621 as template. The fragment was cloned into prokaryotic expression plasmid pET-28a(+) to construct prokaryotic expression plasmid pET28a(+)-BmCPI502. The BmCPI502 gene and BmGAPDH720 gene were amplified by RT-PCR, respectively. The gene fragments of pcDNA3.1(+), BmCPI502 and BmGAPDH720 were digested by double enzyme digestion and ligated with the target gene. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was constructed. The recombinant plasmid was transfected into Hela cells and verified by RT-PCR to obtain the desired target bands. The expression product was detected by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).Results:The recombinant plasmid pET28a(+)-BmCPI502 was obtained, and the 502 bp specific fragment was identified by enzyme digestion, which was in line with the expected value; the eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of the multi-epitope gene was successfully constructed, and the fragment size was in line with the expected value. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was transfected into Hela cells and the recombinant protein was stable expressed. SDS-PAGE analysis showed that the relative molecular weight ( Mr × 10 3) of the recombinant protein was about 50. Conclusions:The eukaryotic expression plasmid of pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi multi-epitope gene is successfully constructed, and the corresponding recombinant protein is obtained in eukaryotic cells. This study has laid a foundation for further study of the purification and biological activity of the recombinant protein.
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To establish an intelligent cervical vertebra maturity assessment system, and to evaluate the reliability and clinical value of the system. Sixty children aged were recruited in the study. Lateral cephalometric radiograph and cone beam CT (CBCT) were taken at the same period. Based on the CBCT data, the system automatically extracted the patient's facial area through Otsu's method, intercepted the sagittal plane by three-dimensional least squares method, captured the second to fourth cervical vertebrae by superpixel segmentation. And then selected points were marked automatically through morphological algorithm and manual method. Consistency test was performed on the two sets of data to compare the reliability of automated cervical morphology capture. According to the parameters of morphological identification, positioning and staging algorithms were designed to form the intelligent cervical vertebra maturity assessment system. The cervical vertebra maturity was also judged manually on the lateral cephalometric radiograph. The weighted Kappa test and the Gamma correlation coefficient were subsequently applied to evaluate the consistency and correlation. The results showed that the cervical vertebra features automatically captured based on CBCT data had a high accuracy on the overall morphological recognition. In the prediction of 8 inflection points out of 13 points, there was no significant difference between automatic and manual method on both X and Y axes (all >0.05). The assessment results of the cervical vertebra maturity of the intelligent system had strong consistency and correlation with the manual recognition results (weighted Kappa value=0.877, Gamma value=0.991, both <0.05). The intelligent cervical vertebrae maturity assessment system based on CBCT data established in this study presents reliable outcome and high degree of automation, indicating that the system may be used clinically.
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Child , Humans , Cephalometry , Cervical Vertebrae/diagnostic imaging , Cone-Beam Computed Tomography , Radiography , Reproducibility of ResultsABSTRACT
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 summarize and analyze clinical characteristics of very elderly patients with acute myocardial infarction(AMI)in order to improve recognization.Methods The clinical materials of 32 very elderly patients with AMI were collected as a observed group from Oct.1999 to Oct.2004,and the clinical materials of 241 patients(aged
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3 mg/L) of hs-CRP (28% vs 0,P
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BACKGROUND AND OBJECTIVES: Early resolution of ST-segment elevation improves the short and long-term mortalities in acute myocardial infarction (AMI). However, the correlations between the ST segment resolution and microvascular integrity, or functional recovery of the left ventricle, were not explored. SUBJECTS AND METHODS: The study population consisted of 42 AMI patients who received thrombolytic therapy (35 male, 54+/-11 years) and consecutive successful percutaneous coronary intervention (PCI) (<30% of residual stenosis, recovered TIMI 3 flow) within 7 days. The coronary flow reserve (CFR) was measured at the segment just distal to the angioplasty site using intracoronary Doppler wire following a successful PCI. Electrocardiograms (ECG) were evaluated before, and within 90-150 minutes after, of the thrombolytic therapy. The percentage change in the ST segment resolution, from the baseline to follow-up, was categorized into complete resolution (70%, n=24), partial resolution (30% to <70%, n=10) and no resolution (0% to <30%, n=8). The ejection fraction (EF) and regional wall motion score indices (RWMSI) were assessed by 2D-echocardiography before, and following the PCI (9+/-5 months). RESULTS: Complete ST segment resolution was observed in 57%, partial resolution in 24%, and no resolution in 19% of patients. The CFR was significantly higher in the complete resolution group than in the no resolution group, and the minimal coronary vascular resistance index was significantly lower in the complete resolution group than in the no resolution group (2.1+/-0.5 vs. 1.4+/-0.4, p=0.006; 2.31+/-0.99mmHg sec cm-1 vs. 3.84+/-2.19mmHg sec cm-1, p=0.035, respectively). The changes in the EF and RWMSI were significantly better in complete resolution group than in the no resolution group (10+/-9% vs. 0+/-5%, p=0.028; -1.03+/-0.50 vs. 0.24+/-0.66, p=0.004, respectively), and the left ventricular end diastolic and systolic volume indices were significantly increased in the no resolution group at follow-up (42.0+/-14.8 mL/m2 vs. 55.8+/-18.1 mL/m2, p=0.006; 23.2+/-10.9 mL/m2 vs. 30.5+/-15.9 mL/m2, p=0.039, respectively). CONCLUSION: In the AMI patients, with a recovered TIMI 3 flow, following thrombolysis and successful elective PCI, there were differences in the coronary flow reserve according to the degree of early resolution of the ST segment. The patients with an early complete resolution of the ST segment showed the most favorable preservation of the microvascular integrities and improvement of the left ventricular function. The degree of early resolution of the ST segment might be a useful indicator for the prediction of left ventricular functional changes at follow-up.