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Objective To optimize the experimental system of dual molecular beacon to rapidly detect My-cobacterium tuberculosis and its resistant strains.Methods Fluorescence quantitative PCR was carried out by selecting different magnesium ion concentration,annealing temperature and primer concentration respectively. Finally,the optimum reaction conditions were obtained.Results In order to ensure the efficiency of amplifica-tion and no non-specific amplification,the final selection of the best conditions were as follows,the concentra-tion of Mg2+was 3.0 mmol/L,annealing temperature was 60 ℃,and the concentration of primers was 0.3 mmol/L.Conclusion The optimal condition of dual molecular beacon experiment was established,which en-sured that the detection of Mycobacterium tuberculosis by molecular beacon quantitative PCR had the advanta-ges,such as simple operation,rapid speed,high sensitivity(the minimum detection limit was 1 CFU/mL)and specificity(only Mycobacterium tuberculosis complex including drug-resistant strains could be detected),good reproducibility(coefficient of variation was < 5%)and other advantages.The study provides the necessary conditions for the dual molecular beacon detection of Mycobacterium tuberculosis.
RESUMO
To investigate the immunological activities of the recombinant human phosphatase D2 (rhPLD2) in vitro and in vivo, especially its ability to reduce inflammatory reactions, the cDNA fragment encoding rhPLD2 was cloned into prokaryotic expression vector pET30a by RT-PCR and the recombinant protein rhPLD2 expressed in E.coli was purified from the inclusion bodies, while the anti inflammatory activity of rhPLD2 was determined by the amount of eosinophils in bronchoalveolar fluid(BALF) and blood and the expression of IL-5 and MMP-9 in lung tissues of guinea pig model of chronic asthma. It was found that the rhPLD2 recombinant protein was obtained from human Daudi cells by cloning to E.coli, which contained no membrane-binding site and signal peptide. The cDNA sequence encoded 631 amino acid residues (GenBank Accession Number: AY178289). The purity of the rhPLD2 approached up to 76% with a bioactivity of 50.9745 units/L (0.9212 g/L). In addition, the anti inflammatory effect of rhPLD2 protein could be demonstrated in the guinea pig model of chronic asthma after treatment with rhPLD2 protein, such as down regulation in the expression of the inflammatory cytokine IL-5. It is concluded that the anti-inflammator activity of the recombinant human truncated PLD2 protein produced from the E.coli plasmid can be demonstrated both in vitro and in vivo.
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The effect of recombinant human phospholipase D2(rhPLD2)in vivo was investigated on the secretion of serum glycosyl phosphatidylinositol-specific phospholipase D(GPI-PLD)in guinea pigs of chronic asthma．Ater treating the guinea pigs attacked by chronic asthma with rhPLD2,the GPI-PLD activity detection was canrried out by phase separation of human placental alkaline phosphatase in Triton X-114．Compared with the healthy guinea pigs(NS group),the serum GPI-PLD in the guinea pigs of chronic asthma are much higher than that of control groups,P≤0．01．Our results showed that rhPLD2 could significantly reduce the secretion of GPI-PLD when the guinea pigs were attacked by chronic asthma．
RESUMO
Objective: To explore the effect of mast cell before or after stimulation of antigen (?IgE HRP).Methods: To observe the shape and calculate the percent of histamine releasing of mast cell used the staindiing method of DAB add neutral red.Results: The tendency of the degranulation of mast cell was declined obviously after adding HUOXIANG ZHENGQI ORAL LIQUOR. Conclusion: HUOXIANG ZHENGQI ORAL LIQUOR can prevent and treat type Ⅰ hyperresponsiveness.
RESUMO
Objective:To study the biologic function of rhPLD2 mutation form.Methods:To adapt the guinea pig chronic asthma model was induced by OVA, the functions of rhPLD2 on PAF was observed through the assay of platelet congregating.Results:rhPLD2 remarkably reduced the lever of PAF in the serum of guinea pig chronic asthma model; compares with the NS group, the P
