| Lipopolysaccharide ( LPS ) are not only the major constituents of the outer membrane of Gram-negative bacteria, but also the toxic determinants for Gram-negative bacteria infection, which is closely related to human health and the development of inflammation. Pattern recognition receptors (PRR) have been considered to be the important initial steps for cellular recognition of LPS and consequent initiation of LPS responses. PRR may be a complex of multiple components. Scavenger receptor , CD 14 , toll-like receptors , B 2-integrins and L-selectin have been shown to be involved in the clearance of LPS or LPS activation. Different PRR transduct LPS signals through different pathways. The process are so complicated that some new ways must be detected to explain the LPS-inflammation courses.lrg means lipopolysaccharide respond gene. Dr Chai Yu-bo carried out PCR-based improved subtractive hybridization,constructed LPS-stimulated human dental pulp cell, and obtained new gene (GenBank: AF143740, etc). The aim of this study is to identify the expressive and contributive rule of human lrg and the function of this protein on cell morphology and cell cycle.Supported by the National Natural Science Foundation of China (No.30170361), We have completed some experiments and got results. Here we report the analysis of the expression pattern and function of human lrg.In this study, the expression pattern of human lrg has been analyzed. At first, human lrg was cloned again and proved correctly. Bioinformatics demonstratd that lrg has 558 base pairs in open reading frame. And L-x(6)- L-x(6)-L-x(6)-L can be found in human lrg sequence.In order to express the recombinant human Lrg protein in E. coli, the encoding region of human lrg cDNA was amplified by PCR, and then subcloned into the prokaryotic expression vector pcTAT to construct pcTAT-hlrg, 6His-TAT was fused with coding sequence of hlrg cDNA in-frame with E.coli BL21 (DE3), And 6His-TAT-hLrg was expressed under IPTG induction, and the expressed products accounted for 51% of the total protein, and it was soluble. The 6His-TAT-hLrg purified through Ni-NTA affinity chromatography column reached electrophoretical pure. In other words, Human Lrg fusion protein was expressed in E.coli successfully with high-efficiency, and Lrg was preliminary purified through Ni-NTA column, Which lays the foundation of human lrg function.To obtain an anti-Lrg immune serum, the fusion proteins were expressed in E.coli and then purified by Ni-NTA column andSDS-PAGE. Rabbits were immunized with preliminary purified Lrg protein, and reinforced three times. Immune serum was collected. At last, immune serum against human Lrg was obtained in immunized rabbits, and its titer was more than 1:1000. The formation of rabbit anti-Lrg immune serum was conformed by ELISA and Western Blot. The purified protein could get across membranes of Dental Papilla Cells within 30 minutes.To analyze the expression level of human Lrg protein, the rabbit anti-Lrg immunol serum has been used as the first antibody. The expression level of human Lrg in several normal fetal tissues was then identified by immunohistochemistry. This assay showed that human Lrg was expressed in almost all tissues detected. The order of positive signal strength was stomach testicle esophagus small intestine liver kidney heart and lung; The coding region of human lrg cDNA was labeled and used to probe human tissues to detect the expression level of human lrg-mRNA. In situ hybridization results demonstrated that human lrg-mRNA was expressed highly in testicle and small intestine.To analyze the intercellular localization of human Lrg, we constructed eukaryotic expressive vector of EGFP-hLrg fusion protein named pIRES2-EGFP-hlrg. This vector was transfected into mouse fibroblast cell line NIH3T3 by way of lipofectin. Fluorescence microscopy analysis revealed that human Lrg localized in cytoplasm mainly. Then the intercellular localization of human Lrg was further analyzed by indirect immunofluorescenc... |
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