[Liangge san effects the expression of CD14 and scaverger receptor in the Kupffer cells of liver of endotoxemia mice].

OBJECTIVE: To study the effects of Liangge San to the expression of CD14 and scaverger receptor(SR) in the kupffer cells of liver and the pathological changes of liver tissue of endotoxemia-mice. METHOD: The model was established with intravenous injection of lipopolysaccharide (LPS) and at the same time different dose Liangge San were given. The expression of CD14 and scaverger receptor were detected with immunohigtochemistry at the 2nd, 4th, 8th hour ofter injury and analyzed with computer image system, and the pathological changes of liver tissue were also observed. RESULT: At the three different hours, the expression of CD14 and scaverger receptor in macrophages of liver of LPS-injury group showed significant increase and significant decrease respectively, compared with that of the blank-control group (P < 0.01). The expression in dexamethasone group and Liangge San different dose groups were intermediate between those in injury group and those in control group. Compared with expression of LPS-injury group, those of dexamethasone group and Liangge San different dose groups showed significant differences (P < 0.01), especially that of Liangge San high dose group. Liver cells showed vacuole change. Changes of CD14 and SR expression were paralleled with the severity of liver damages of the mice. CONCLUSION: Liangge San can inhibite the up-regulation of CD14 expression and down-regulation of scaverger receptor expression in a dosage-dependent manner and also alleviate the damages of liver induced by LPS.

[Construction and expression of green fluorescent protein fusion vector of p38 regulated/activated protein kinase].

OBJECTIVE: To construct the expression vector of p38 regulated/activated protein kinase (PRAK) fused to green fluorescent protein which is capable of expression in mammalian cells. METHODS: PRAK with His-tag in pET-14b expression vector was subcloned into the green fluorescent protein vector pEGFP-C2. The recombinant vector was then transfected into HeLa cells, followed by observation of the cells with fluorescent microscope. RESULTS: Identification by enzyme digestion, PCR and sequencing confirmed successful construction of the recombinant vector, which was highly expressed in HeLa cells. Green fluorescence of the fusion protein EGFP-PRAK was observed mainly in the cell nuclei. CONCLUSION: The expression vector of PRAK fused to green fluorescent protein is successfully constructed and expressed in mammalian cells, which may facilitate the study of intracellular localization and translocation of PRAK.

[Construction of GFP-AWP1 fusion gene vector and its expression in 293 cells].

OBJECTIVE: To construct green fluorescent protein (GFP)-AWP1 (a novel human protein associated with protein kinase C-related kinase 1) fusion gene vector for observing the expression and localization of AWP1 in 293 cells. METHODS: The coding region in AWP1 cDNA was amplified by RT-PCR from human endothelial cell line ECV304 and recombined into pEGFP-C2 plasmid expressing GFP. After identification with restriction endonucleases and sequence analysis, the recombinant plasmid was transfected into 293 cells with the cationic liposome DOTAP as the transfection reagent. The expression and localization of AWP1 were observed under a fluorescence microscope. RESULTS: Restriction endonuclease assay and sequence analysis verified the successful construction of the recombinant vector pEGFP-C2/AWP1, and GFP-AWP1 fusion protein was highly efficiently expressed in 293 cells. Under fluorescent microscope, green fluorescence was seen homogeneously distributed in the entire cell body of the cells transfected by the empty vector pEGFP-C2, but diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C2/AWP1. CONCLUSION: GFP-AWP1 fusion gene vector is successfully constructed and the fusion protein expressed in the cytoplasm of 293 cells.

[Construction and expression of the fusion vector of His-tagged human ARPC2 gene].

OBJECTIVE: To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purification in E. coli. METHODS: ARPC2 cDNA codon region was amplified by PCR from human liver cDNA library and cloned into pET-14b vector following the routine procedures. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 (DE3) competent cells, and the expression of His-ARPC2 fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography. RESULTS: The constructed His-ARPC2 fusion protein vector was highly efficiently expressed in E. coli. With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 000 was obtained. CONCLUSION: The expression vector of His-ARPC2 fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the physiological functions of ARPC2 and characterization of its interaction proteins.

[Vector construction for the intracellular domain of human receptor for advanced glycation end- products and expression of the fusion protein].

OBJECTIVE: To construct a eukaryotic expression vector for GST-tagged intracellular domain of human receptor for advanced glycation end-products (hRAGE), and to study the function of the expressed fusion protein and identify its interacting proteins. METHODS: The coding sequence of the intracellular fragment of hRAGE was amplified by PCR and inserted into pGEX-KG vector, a general GST fusion protein expression vector. After PCR identification, endonuclease digestion and DNA sequencing, the recombinant was transformed into E.coli BL21 to achieve the expression of GST fusion protein induced by isopropyl-beta-D-thiogalactoside (IPTG), followed by purification of the protein on glutathione-superflow resin. RESULTS: The recombinant of GST/hRAGE-C was constructed and identified by PCR, endonucleases digestion and DNA sequencing. After protein expression was achieved in E. coli, a molecular mass of 35 kD GST fusion protein was purified, whose molecular mass and purity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). CONCLUSION: The expression vector for intracellular domain of hRAGE has been successfully constructed and efficient expression of the fusion protein is obtained, which can be of value for further studies.

[Screening and identification of proteins interacting with p38 MAP kinase via T7 phage-display screening system].

OBJECTIVE: To screen and identify proteins that interact with p38 mitogen-activated protein (MAP) kinases by means of T7 phage-display screening system. METHOD: His-tagged fusion protein of p38 MAP kinase was used to coat a 96-well ELISA plate and Ni-NTA resin, which served as the media for screening human lung and liver T7 phage cDNA libraries. RESULTS: After 4 rounds of biopanning, 86 independent plaques were selected and processed by EDTA. The inserted gene fragments from these plaques were amplified by PCR, the products purified by a gel recovery method. The sequences of the insertions were identified and analyzed with BLAST program in GenBank. Forty-six clones were found to encode proteins. CONCLUSION: T7 phage-display screening system is convenient, rapid and effective for screening the P38 MAP kinase-binding proteins.

[Construction of vector of his-tagged cytoplasmic fragment of human toll like receptor 4 and its expression in E.coli].

OBJECTIVE: To construction of vector of his-tagged cytoplasmic fragment of human Toll like receptor 4 (hTLR4) and its expression in E.coli. METHODS: hTLR4 cytoplasmic cDNA codon domain was amplified by polymerase chain reaction (PCR) and cloned into pET-DsbA2.0 plasmid expressing His-DsbA fusion protein. After being identified by the assay of restrictional enzyme and sequencing, His-Dsb A fusion proteins were induced with isopropy-beta-D-thiogalactoside (IPTG) and further purified. RESULTS: A fusion protein with molecular weight of 42 kd was obtained. CONCLUSION: hTLR4 which was constructed and expressed successfully under nondenaturing conditions provides a tool for further studies.

Regulation of inducible nitricoxide synthase gene transcription by p38 mitogen-activated protein kinase in human embryonic kidney 293 cells.

OBJECTIVE: To study the transcriptional regulation of inducible nitric oxide synthase (iNOS) gene by p38 mitogen-activated protein kinase (MAPK). METHODS: With human embryonic kidney (HEK) 293 cells as the target and the assistance of lipofectamine-mediated co-transfection techniques and luciferase reporter gene systems, FLAG-tagged p38 isoforms (namely FLAG-p38 alpha, FLAG-p38 beta;, FLAG-p38 gamma and FLAG-p38 phi;) in pcDNA3, pcDNA3, piNOS-Luc and pCMV-beta; were transfected into HEK 293 cells, and the relative activity of luciferase was subsequently tested. RESULTS: Highest luciferase activity occurred only in p38 alpha group compared with the other three isoform groups under no stimulation. Under the stimulation by lipopolysaccharide (LPS), the luciferase activity of each group was obviously increased and the highest activity occurred in p38 beta group. CONCLUSION: LPS can induce transcription and activation of iNOS gene, and p38 MAPK is involved in the transcription regulation of iNOS gene in HEK 293 cells.

Role of p38 mitogen-activated protein kinase in lipopolysaccharide-induced expression of inducible nitric oxide synthase in human endothelial cells.

OBJECTIVE: To understand the role of p38 mitogen-activated protein kinase (p38MAPK) in the expression of inducible nitric oxide synthase (iNOS) and NO production in human endothelial cells under the stimulation by lipopolysaccharide (LPS). METHODS: NO level in the supernatant of the cell culture media was measured with Griess method, and iNOS protein and mRNA expressions by the cells were detected with immunofluorescence analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. Immunoprecipitation assay was employed to examine p38 MAPK activity. RESULTS: It was shown that in comparison with the basal level of iNOS expression in cultured endothelial cells line ECV304, iNOS mRNA and protein expressions were significantly increased in the cells after LPS stimulation. In response to LPS treatment, obvious enhancement of p38 MAPK activity in ECV304 took place after the stimulation, with the peak level occurring at 15 min that maintained for approximately 45 min before gradual decline. When treated with SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK, significant inhibition of LPS-induced iNOS protein and mRNA expressions was observed. CONCLUSIONS: p38 MAPK plays an important role in iNOS expression and NO production in ECV304 cells, and, inhibition of the signal transduction pathway may consequently be an effective approach to reduce the production of iNOS and other cytokines for the treatment of septic shock.

[Construction and expression of red fluorescent protein fusion vector incorporating p38 mitogen-activated protein kinase].

OBJECTIVE: To construct the vector that expresses the fusion protein of p38 mitogen-activated protein kinase (MAPK) and red fluorescent protein (RFP) in mammalian cells. METHODS: FLAG-tagged p38 MAPK in pcDNA3 vector was subcloned into RFP vector pDsRed1-N1, the construct of which was then transfected into HeLa cells and observed with fluorescence microscope. RESULTS: The recombinant plasmid was verified by enzyme digestion, PCR and sequence analysis, and p38 MAPK-RFP fusion protein was highly expressed in HeLa cells. Fluorescence microscope found the red fluorescence distributed all over the cytoplasm and in the nuclei as well. CONCLUSION: The expression vector for p38 MAPK-RFP fusion protein is successfully constructed and effective expression of this fusion protein is achieved, which might be instrumental in the study of intracellular localization of p38 MAPK.