iASPP monoclonal antibody and its research applicationObjective:To prepare the mouse anti-human iASPP monoclonal antibody by single-shoti" /> Dissertation > Excellent graduate degree dissertation topics show

iASPP and application of monoclonal antibodies and iASPP new spliceosome (iASPP-SV) inhibition of p21 ~ (WAF1/CIP1) transcription mechanism

Author: DiaoShiYong
Tutor: WangJianXiang;WangMin
School: Peking Union Medical College , China
Course: Internal Medicine
Keywords: iASPP iASPP-SV DNA immunization monoclonal antibody ASPP iASPP p21WAF1/CIP1 Sp1
CLC: R392
Type: PhD thesis
Year: 2008
Downloads: 107
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Abstract


PartⅠPreparation of anti-human oncoprotein ASPP' dissertation">iASPP monoclonal antibody and its research applicationObjective:To prepare the mouse anti-human iASPP monoclonal antibody by single-shoting plasmid DNA intrasplenic immunization and identify its biological characteristics.Methods:The cDNA of human iASPP-SV was cloned by reverse transcriptase polymerase chain reaction(RT-PCR) from U937 cell line.The full-length cDNA of iASPP-SV was cloned into the eukaryotic expression vector pcDNA3.1(+) and the full-length cDNA or small fragment of iASPP-SV was cloned into the prokaryotic pET28a(+).The full-length or small fragment peptide of iASPP-SV with His-tag(PIAS and PIAF) was induced to express PIAS and PIAF proteins in E.coli Rosseta(DE3) strain by inclusion body, respectively.The PIAS and PIAF inclusion bodies were denatured by guanidine hydrochloride and were purified by by only one step of Ni2+ NTA chromatography.After 3 days injecting the plasmid pcDNA3.1-iASPP(CIAF) into spleens of Balb/c mice once,the spleen cells were fused with NS1 cells.Then monoclonal antibodies were prepared by classical hybridoma technique.We used ELISA method to scan the fused clones by using the full length and small fragment of his-tag fusion iASPP-SV protein expressed by the prokaryotic cells.And we identified some biological functions of the antibodies produced by the positive subclones.Results:We cloned the cDNA of iASPP-SV by RT-PCR from U937,and succeeded in constructing PIAF,PIAS and CIAF expression vector.The recombinant vector can express the small fragment and full length of iASPP-SV fusion proteins in E.coli Rosetta(DE3) strain induced by IPTG. The products were identified as by SDS-PAGE and Western blot.We purified the fusion protein by only one step of Ni2+ NTA chromatography,and the purity of the fusion protein was over 80%analyzed by SDS-PAGE.We prepared 10 hybridoma cell lines,which can secrete the antibodies against human iASPP proteins.And the antibodies can be applied in western blot、immunohistochemistry.Conclusions:In tis study we prepared the mouse anti-human oncoprotein iASPP monoclonal antibodies by single-shoting plasmid pcDNA3.1-iASPP-SV DNA intrasplenic immunization successfully.The antibodies we prepared can identify the two isoform of iASPP:iASPP-SV and iASPP specifically. We founded the excellent base for the future works on examination of iASPP protein and its function and mechanism. PartⅡiASPP-SV interacts with Sp1 and represses p21WAF1/CIP1 transcription mediated by Sp1Objective:To investigate the interaction of iASPP-SV and transcriptional factor Sp1,the influence and mechanism of iASPP-SV on p21WAF1/CIP1 promotor mediated by Sp1.Methods:We transfected the iASPP-SV and Sp1 into 293 and KB cells to find that whether iASPP-SV can interact in vivo and co-locate in nuclear. We further studied the the influence and mechanism of iASPP-SV on p21WAF1/CIP1 promotor mediated by Sp1 by using luciferase reporter and chromatin immunoprecipitation technologies.Results:We found that iASPP-SV could interact with Sp1 in vivo and co-locate in nuclear,iASPP-SV can inhibit the transcriptional activities of pGL3-Basic-p21WAF1/Cip1-promoter and p21WAF1/Cip1-101-promoter luciferase reporter.And the further study showed iASPP-SV can bind Sp1 binding site on p21WAF1/Cip1 promoter.Conclusions:iASPP-SV can interact with Sp1 in vivo and co-locate in nuclear;iASPP-SV can inhibit the transcriptional activities of p21WAF1/CIP1 promotor mediated by Sp1 and bind Sp1 binding site on p21WAF1/Cip1 promoter.

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