ABSTRACT
Objective: To investigate changes of the endothelin-1(ET-1) and its receptor (endothelin receptor subtype A, ET AR ) mRNA expression in some organs(kidney, lung and small intestinal mucous membrane) in the sepsis and septic shock rats. Methods: Twenty-four male rats randomized into sepsis group, septic shock group, control and normal group was infused with endotoxin(LPS) via indwelling right atria catheters except normal and control group. RT-PCR was used to detect kidney, lung and small intestinal mucous membrane tissue mRNA expression of the ET-1 , ET AR and glucose-6-phospho dehydrogenase(G-6-PD) in every group.Serum BUN, Cr, ALT and A were determined. The arterial oxygen tension (PaO 2) and arterial carbon dioxide partial pressure(PaCO 2)were measured in every group. Results: ET-1 mRNA and ET AR mRNA expression in the sepsis group and septic shock group were significantly higher than in normal group. There were significant differences between the normal/control group and sepsis/shock group in BUN,Cr,ALT,PaO 2 and PaCO 2. Conclusion: A higher expression of ET-1 mRNA and ET AR mRNA may be one of the startup factors on multiple organ dysfunction syndrome (MODS) and may play an important role on pathogenesis in sepsis and septic shock.
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Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko-expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector-pEF-GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV-GFP with CMV promoter and by pdCMV-GFP with double copies of CMV-GFP expression unit. There was no significant difference between the colonies transfected by pCMV-GFP and pdCMV-GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.
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ObjectiveTo construct the eukaryotic coexpression plasmid CEA/IL-2, and to lay the foundation for further studying CEA nucleiotide vaccine , adjuvant and their effects of special antitumor immunity.MethodsThe eukaryotic expression plasmid (pcDNA3-CEA)containing the gene coding for CEA was obtained by RT-PCR and gene recombination techniques.Using enzymolysis,ligation and other techniques,an eukaryotic coexpression plasmid (pIRES-CEA/IL-2)containing two expression unites of CEA and IL-2 gene connected with internal ribosome site was constructed.ResultsThe coexpression plasmids were transformed into COS7 cells and expression of two proteins were demonstrated by ELISA, and flow cytometer and elecsy.CEA and IL-2 were (23.73?0.26)ng/ml,and(20.17?0.13)ng/ml respectively.ConclusionsThe eukaryotic expression plasmids pIRES-CEA/IL-2 could be successfully constructed and transformed into COS7 cells.Expression of two proteins were demonstrated with no difference on expression.
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Objective: To screen out DNA binding proteins specifically recognizing CpG immunostimulatory sequence (ISS) for further investigating the molecular mechanisms of ISS. Methods: Yeast one hybrid system was adapted in screening a human bone marrow cDNA library using 4 copies of ISS as bait. The ISS binding activity of the positive clone was confirmed by electrophoretic mobility shift assay (EMSA). Results: Four dual positive colonies were obtained, two of them encoded proteins with unknown functions. The other 2 encoded light chains of immunoglobulin with amino sequences homology to anti DNA Ab and HBsAb respectively. EMSA showed HBsAb specifically bound to CpG ODN at pH6.4 and pH 5.8. Conclusion: HBsAb may have ISS specific DNA binding activity.
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Objective: To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin neuraminidase(HN) of Newcastle disease virus (NDV), and to study its mechanism and value in antitumor therapy. Methods: The HN cDNA was abtained from NDV with RT PCR and an eukaryotic expression vector of HN gene ( pcDNA3 HN ) was constructed. The antitumor effect was evaluated after injecting pcDNA3 HN into mice bearing B16 melanoma. Results: The HN cDNA of NDV was successfully cloned and pcDNA3 HN had a good expression in COS 7 cells. Animal experiments suggested that the pcDNA3 HN could significantly increase CTL and NK activity of tumor bearing mice. Conclusion:The eukaryotic expression plasmid containing the gene coding for the (HN) has the function of increasing CTL and NK activity of tumor bearing mice.