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The Expression of the PLA2 Domain of the Periplenta Fuliginosa Densovirus Structural Protein Gene in E. Coli and Preliminary Study of Its Function

Author: YuHaiYang
Tutor: HuYuanYang
School: Wuhan University
Course: Microbiology
Keywords: The fuliginosa densonucleosis virus Phospholipase A2 The prokaryotic expression Affinity chromatography
CLC: Q786
Type: Master's thesis
Year: 2005
Downloads: 77
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Abstract


Fuliginosa the main cockroach species distribution in urban and rural indoor , the fuliginosa densovirus (Periplaneta fuliginosa Densovirus, PfDNV) the cockroaches densovirus a classification and identification at home and abroad . Physical and chemical properties of its virus genome structure analysis , and histopathological aspects of the study have been completed . But for a long time, due to the lack of a stable PfDNV vitro infection cell model , function of PfDNV structural proteins , the molecular mechanism of its replication infection remains unclear . Study found that parvovirus structural protein having phospholipase A2 (PLA2) motif secretory phospholipase motif (eg : venom and bee venom , etc. ) have a high homology , including the conserved calcium binding to point YXGXG and catalytic active sites HDXXY . The indel mutation that the functional domains play a regulatory role in the infection process of the virus particles . Accordingly, in order to further research the fuliginosa densovirus phospholipase A2 functional domains was amplified by polymerase chain reaction (PCR) the functional domains pieces to get PfDNV containing phosphorus lipase A2 (PLA2) , the connection into pMD18-T vector and subcloned into the prokaryotic expression vector pET28a and pET26b build the correct reading frame recombinant expression vector pET28a-PLA and pET26b - PLA , transformed E. coli BL21 - codonplus ( DE3 ) -RIL induced by IPTG , SDS -PAGE get the purpose of fusion protein , pET28a-PLA general conditions induced from inclusion bodies , pET26b-PLA through optimization of expression conditions in the cytoplasm and periplasm soluble recombinant protein expression in the form to Ni-NTA affinity and chromatography column purified under non - denaturing conditions of the target protein , lecithin as substrate measured phospholipase A2 activity , preliminary comparative analysis of the results PfDNV results successfully expressed recombinant protein of PLA2 these initial work for the next antisera Preparation of PfDNV functional domains of phospholipase A2 virus particles exist in the form and through methods such as site-directed mutagenesis laid the foundation for its in-depth study of the biological characteristics .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering) > Gene expression
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