Conventional reverse transcriptase polymerase chain reaction (RT-PCR) and optimized of a closed tube reverse-transcription loop-mediated isothermal amplification (RT-LAMP) were used for detection of pandemic (H1N1) 2009 virus and the optimized of a closed tube RT-LAMP methods were compared with the conventional RT-PCR with respect to specificity and sensitivity. In this study, optimized RT-LAMP detected 2 copies of target RNA by visual detection with modified dye. Reaction time, temperature and quantity of each reagent were optimised for the detection of the virus. The sensitivity of detection limit by optimised RT-LAMP was 100 times as that of conventional RT-PCR. Amplification of DNA can be identified by visualization with modified dye, which reduces the cross-contamination caused by opening tube. The sensitivity of visual detection was equivalent to that of electrophoresis analysis. Additionally, the method was specific as no cross-reaction was observed among samples from human blood, Escherichia coli and other related viruses including human seasonal influenza A, subtypes H1N1, H1N2 and H3N2 viruses. These results demonstrate that the optimized RT-LAMP assay for pandemic (H1N1) 2009 virus RNA was a valuable tool with simplicity, rapidity and specificity, as well as its superiority for the screening and surveillance of influenza in developing countries.
Key words: Pandemic (H1N1) 2009 virus, loop-mediated isothermal amplification (LAMP), reverse transcriptase polymerase chain reaction (RT-PCR), HA gene.
Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation.
Both interleukin-1 and IgE are important in the pathogenic mechanism of the allergy asthma. cDNA of interleukin-1receptor antagonist (IL-1ra) and IgE were cloned and a prokaryotic expression vector IL-1ra-Fcepsilon/pBV220 was constructed. The vector was transformed into Escherichia coli BL21(DE3). The fusion protein was expressed successfully in the form of inclusion body. The recombination protein of IL-1ra-Fcepsilon was highly purified by chromatography of gel filtration and ion exchange, which was identifited by Western blotting. The cell assay showed that the activity of IL-1ra-Fcepsilon was as high as IL-1ra in vitro after refolding. The pharmacokenetic profile of IL-1ra-Fcepsilon and L-1ra was analyzed, and the half time of IL-1ra-Fcepsilon is 4.78 times than that of IL-1ra.
After amplifying the SAK coding sequence from the chromosomal DNA of the lysogen of Staphylococcus aureus strain FR610, the isolated fragment was sequenced by dideoxy method, recombined in vitro with plasmid pBV220, and transformed E. coli DH5alpha cells. An high-expression strain was obtained. The expressed SAK amounted to 70% of the total bacterial protein. The specific activity of the SAK was 3×10~(6) units/mg, measured by comparison with a standard preparation. A high-expression E. coli strain for SAK was successfully obtained.
Objective To study the effects of interleukin 1 receptor antagonist (IL-1ra) on allergy asthma and its mechanism. Methods Thirty female SD rats underwent intraperitoneal and hypodermic injection of ovalbumin (OVA) on days 1 and 14, and then underwent spraying of OVA aerosol since day 21 for7 days so as to'provoke asthma, and then the rats were randomly divided into 3 equal groups: asthma model group, low dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 6 mg/kg before each provocation (low dose treatment group), and high dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 30 mg/kg before each provocation(high dose treatment group). Another 10 rats were used as normal controls. Twenty-four hours after the last provocation physiological monitoring equipment was used to detect the pulmonary function. Then the rats were killed. Bronchoalveolar lavage fluid (BALF) was collected. ELISA was used to detect the serum IgE content. The ratio of inflammatory cells from the BALF was calculated. Microscopy was conducted to observe the histopathology of lung. RT-PCR was used to examine the mRNA expression of NF-κB and signal transducer and activator of the transcription 6 (STAT6). Results The respiratory rate, expiratory flow, percentage of eosinophils in BALF inflammatory cells, peripheral blood IgE concentration, mRNA expression of STAT6 and NF-κB of the asthma group were (206±11) times/min, (77±8 ) μl/s, 24.8% ± 1.3%, (72.5 ± 8.1) ng/ml, 0.294 ± 0.048, and 0.686 ± 0.052 respectively, all significantly higher than those of the low dose treatment group [ (183 ± 9) times/min, (64±5) μl/s, 18.5%±3.1%, (63.4±4.8) ng/ml, 0.229±0.038, and 0.613±0.062 respectively, all P＜0.05] and those of the high dose treatment group [ (181 ± 11) times/min, (57 ± 4) μl/s, 14.7% ±2.1%, (41.4±7.4) ng/ml, 0.194±0.076, and 0.352±0.267, all P＜0.05]. The therapeutic effect of high dose treatment group is superior to that of low dose treatment group (P＜0.05). Conclusion IL-1ra is significantly effective in treatment of allergic asthma, and its potential mechanism is through regulating both STAT6 mRNA and NF-κB mRNA expression simultaneously.
Key words:
Sthma; Receptors, interleukin-1 ; Intracellular signaling peptides and proteins; NF-Kappa B
The aim of this study was to realize the oral delivery of SAK-HV protein and improve its oral bioavailability based on chitosan quaternary ammonium salt-PLGA microsphere. The results showed that the SAK-HV-loaded microsphere can overcome the multiple obstacles for oral adsorption and adhere effectively to the jejunal segment of a rat. The pharmacokinetic analysis of the oral drug-loaded microspheres in rats showed that the blood drug concentration of SAK-HV reached the peak value at 4 h after oral administration, and the relative oral bioavailability of SAK-HV was 3.4%. Additionally, after oral administration to the mice, a higher level of antibody against SAK-HV was produced on day 21 compared with that in the control and injection groups, and the antibody titre was 7.2 times that of the tail vein group. This work suggests that the microsphere of the chitosan quaternary ammonium salt-PLGA may be a promising drug delivery system for the oral administration of SAK-HV protein.
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