iASPP单克隆抗体的制备与应用及iASPP新的剪接体（iASPP-SV）抑制p21^WAF1/CIP1转录机制研究

【作者】 刁世勇；

【导师】 王建祥； 王敏；

【作者基本信息】 中国协和医科大学 ， 内科学， 2008， 博士

【摘要】 第一部分抗人iASPP单克隆抗体的制备及研究应用实验目的:应用脾脏内注射真核iASPP-SV表达质粒免疫的方法制备抗人iASPP单克隆抗体（McAb）,并对其生物学特性进行初步研究。实验方法:RT-PCR方法克隆iASPP-SV编码区cDNA。将扩增所得到的全长基因克隆到真核表达载体pcDNA3.1（+）中,并将iASPP-SV基因全长和基因片段克隆到原核表达载体pET28a中。诱导PIAF和PIAS载体在大肠杆菌Rosseta（DE3）中表达PIAF和PIAS蛋白。通过盐酸胍变性溶解包涵体,并经镍柱纯化得到重组PIAF和PIAS蛋白。采用脾脏内注射真核pcDNA3.1-iASPP-SV（CIAF）免疫Balb/c小鼠3天后,取小鼠脾脏与小鼠骨髓瘤细胞（NS1）用传统的淋巴细胞杂交瘤技术制备抗人iASPP单克隆抗体。应用原核表达的iASPP-SV全长及iASPP-SV片段His-tag融合蛋白进行间接ELISA法筛选阳性克隆。并对亚克隆后的阳性杂交瘤细胞株所制备的抗体的生物学功能进行了初步鉴定。实验结果:我们利用RT-PCR方法从人白血病细胞系U-937中克隆出癌基因iASPP-SV编码cDNA,成功构建iASPP-SV表达载体PIAF、PIAS和CIAF,重组质粒读码框和序列与预期一致。在IPTG诱导下,重组载体大肠杆菌Rosetta（DE3）菌株,表达产物经SDS-PAGE和western blot方法分析证实系PIAS和PIAF融合蛋白。利用Ni²⁺鳌合层析的方法纯化PIAS和PIAF融合蛋白,经SDS-PAGE鉴定其纯度超过80%。用CIAF质粒通过脾脏内注射免疫所获得的小鼠脾脏细胞通过细胞融合、筛选和克隆化培养最后得到10株单克隆抗体细胞株。我们所获得的抗体能与天然iASPP-SV和iASPP蛋白及重组iASPP-SV蛋白特异性结合,而且实验表明所获得的抗体可以应用于western blot、免疫组织化学等实验。实验结论:本研究成功地应用脾脏内注射真核iASPP-SV表达质粒免疫制备了针对人iASPP蛋白的抗体,该抗体能够特异识别iASPP-SV和iASPP两个异构体。为进一步对iASPP蛋白水平检测以及其功能、作用机制等实验研究奠定了良好的工作基础。第二部分iASPP-SV能够与Sp1相互作用并抑制Sp1介导的p21WAF1/CIP1转录研究目的:探讨iASPP-SV与转录因子Sp1相互作用和对Sp1介导的p21^WAF1/CIP1启动子转录的影响及其作用机制。研究方法:将FLAG-iASPP-SV与Sp1质粒共转染293和KB细胞系应用免疫共沉淀、免疫荧光方法检测iASPP-SV能否与Sp1相结合及其在细胞核内共定位。应用荧光素酶报告基因和染色质免疫沉淀技术进一步深入研究iASPP-SV对Sp1介导的p21^WAF1/CIP1启动子的转录影响及其机制。结果:我们将iASPP-SV与Sp1共转染细胞后发现iASPP-SV能够在细胞内与Sp1相结合,并共定位于细胞核内。iASPP-SV可以抑制含有Sp1 1-6结合位点的pGL3-Basic-p21^WAF1/Cip1-promoter以及仅含有Sp1 3-6结合位点的pGL3-Basic-p21^WAF1/Cip1-101-promoter荧光素酶报告质粒的转录活性。进一步研究发现iASPP-SV可以结合于p21^WAF1/Cip1启动子区的Sp1结合位点。结论:iASPP-SV可以在细胞内结合Sp1并共定位于细胞核;iASPP-SV可以抑制Sp1介导的p21^WAF1/Cip1转录;iASPP-SV可以结合于p21^WAF1/Cip1启动子区的Sp1结合位点。更多还原

【Abstract】 PartⅠPreparation of anti-human oncoprotein 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 p21^WAF1/CIP1 transcription mediated by Sp1Objective:To investigate the interaction of iASPP-SV and transcriptional factor Sp1,the influence and mechanism of iASPP-SV on p21^WAF1/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 p21^WAF1/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-p21^WAF1/Cip1-promoter and p21^WAF1/Cip1-101-promoter luciferase reporter.And the further study showed iASPP-SV can bind Sp1 binding site on p21^WAF1/Cip1 promoter.Conclusions:iASPP-SV can interact with Sp1 in vivo and co-locate in nuclear;iASPP-SV can inhibit the transcriptional activities of p21^WAF1/CIP1 promotor mediated by Sp1 and bind Sp1 binding site on p21^WAF1/Cip1 promoter.更多还原