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Polyclonal Antibody Specific Identification、Expression and Location of Allantoicase(AllC) from Dictyostelium Discoideum

Author: ChenNengXing
Tutor: HouLianSheng
School: East China Normal University
Course: Zoology
Keywords: Dictyostelium discoideum allantoicase polyclonal antibody Western blotting immunofluorescence
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 39
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Abstract

The social amoeba Dictyostelium discoideum which phagocytosis soil bacteria for food is a unicellular eukaryote.Under favorable condition,it multiplies as a unicellular organism.Upon starvation,a pathway involving aggregation,mound,slug, culmination stages induces the formation of a fruiting body consisting of a head of spores supported on a stalk of vacuolated cells.Spores await dispersal and germinate in favourable conditions for amoeboid growth.A wide range of fundamental cellular processes are common to D.discoideum and metazoans.These processes include cytokinesis、motility、phagocytosis、chemotaxis、signal transduction and aspects of development such as cell sorting、pattern formation and cell-type determination. Dictyostelium discoideum is an excellent system in which to study cell mobility,cell-cell signal transduction,cell type differentiation,development and so on for its unicellular and multicellular life stages and rich life phenomena.gp150 is encoded by lagC gene,and it is a membrane glycoprotein.High concentrations of gp150 are present at cell-cell contacts,they regulate cell-cell adhesion by heterophilic interactions,gp150 is expressed on cells in the early post-aggregation stage,it can be detected in the wild type KAx-3 cells but not in the mutant type AK127 cells by Western blot.The mutant type strain AK127,as a result of absence of gp150,can’t complete multicellular development and the development is arrested at the loose aggregate stage.The obtained data suggested gp150 proteins play an important part in Dictyostelium discoideum development,it may take part in the signaling pathway for regulating cell differentiation and cell apoptosis.So far,our knowledge of the signaling pathway is still very limited. Allantoicase is an important enzyme in the degradation of purines.It catalyzes the conversion of allantoic acid into ureidoglycolate and urea.And the former was resolved into ammonia、CO2、glyoxylate.According to the research discovery,the different species allantoicase gene all is located in the chromosome,but expresses the oprotein locates differently in the cell;The different living creature purine metabolism end product is dissimilar,therefore in pathways each kind of enzyme activity is dissimilar,in the majority fish and the amphibians the allantoicase has activity,in reptiles,birds and the mammal does not have aUantoicase activity,however in the mouse,mammal,and human organizations has the allantoicase gene duplication and expression.Why can allantoicase activeness vanish gradually in the biological evolution process? Why in higher mammalia doesn’t have the allantoiase enzyme activity but have the allantoicase gene expression actually? Its mechanism still not clear,this caused us enormous interest in the allantoicase research.But in the Protozoa Dictyostelium discoideum about the localization of allantoicase and the molecular signal conduction mechanism are still not clear.So we use the allantoicase which is obtains by our laboratory to prepare the polyclonal antibody,The titer of the antiserum measured by ELISA could achieve 1:64000.;Western blotting examinate anti-body specificity and appetency,the antiserum can specifically combine with goal protein,compared serum is not have the mixed with antigen.Using the obtained anti-body analysis allantoicase in cell expression situation by Western blot,found that allantoicase have expression in all multicellular development.Aggregation stage the protein gray value of allantoicase is about 40-50,in the stage of mound and slug allantoicase expression have a significant increase and gray value is 104-109.Thats mean in the stage of mound and slug allantoicase expression quantity is double than aggregation.prompts that the allantoicase play a important role in multicellular development and differentiation of KAx-3 cells;at the same time found that its still having 62kD protein expression by allantoicase polyclonal antibody in multicellular development.The expression quantity is higher than allantoicase but the protein expression change trend is similar to allantoicase.suggesting that allantoicase may have some relations with 62kD protein.Analysising the location of allantoicase in Dictyostelium discoideum cell through the immunity fluorescence technology and the laser,discovered the allantoicase expression in prestalk cells is higher than prespore cells,suggest allantoicase may have a role in differentiation of prestalk cells;fluorescence also found that allantoicase in the membrane fluorescence be enhanced and found Capping of allantoicase,Capping structure is a typical characteristic of membrane protein.suggesting that allantoicase may play a role with membrane-associated protein of Dictyostelium discoideum.By the analysis of molecular biology softwares found that allantoicase have no transmembrane-domain but have high expression of allantoicase around the cell membrane.We analyzed the allantoicase may be anchoring with the cell membrane protein through connexin and then play a important role in multicellular development of Dictyostelium discoideum.allantoicase is found by members of my laboratory through DDRT-PCR of wild type KAx-3 cells and mutant type strain AK127(knockout lagC gene),so research might be considered as the background for the research on intercellular signaling pathway involving adhesion molecule gp150 in Dictyostelium discoideum, as well as favor the further study on regulation of signaling transductions and their relationships in other more advanced organisms.

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