【作者】 李婧；

【导师】 郭风劲；

【作者基本信息】 重庆医科大学 ， 遗传学， 2009， 硕士

【摘要】 研究目的真核细胞中X盒结合蛋白1(X-box binding protein 1, XBP1)作为一种转录活化因子发挥着多种不同的功能,具有广泛的生物学效应,参与机体的多种生理及病理过程,其对疾病发生及发展的影响是近年来研究的热点。本研究首先运用多聚酶链式反应(PCR)、基因克隆等分子生物学技术构建了重组质粒pET32a-XBP1u,通过将其转化入大肠杆菌E.coli BL2l,得到大量包涵体表达的目的蛋白XBP1u,利用Ni2+-NTA树脂对目的蛋白进行纯化,之后进一步应用Western blot对纯化的目的蛋白进行分析,以鉴定得到的纯化XBP1u蛋白,用该纯化蛋白免疫家兔制备抗XBP1u多克隆抗体,从而为进一步研究XBP1u的生物学功能及其他相关后续研究奠定基础。研究方法(1)使用总RNA提取试剂盒(TRIZOL试剂盒)从肝癌细胞(HepG2)中提取总RNA,并利用RT-PCR两步法扩增出目的片段XBP1u。(2)利用分子克隆技术将目的基因克隆到pGEM-T载体上,构建成重组载体pGEM-T-XBP1u。(3)利用分子克隆技术构建原核表达载体pET32a-XBP1u。(4)将重组载体pET32a-XBP1u转化入大肠杆菌E.coli BL2l,用IPTG诱导转化菌大量表达,得到目的蛋白XBP1u,SDS-PAGE分析其表达形式,并利用Ni2+-NTA树脂对目的蛋白进行纯化,之后用BCA法测定所表达的目的蛋白浓度。(5)利用Western blot鉴定目的蛋白的正确性。(6)用纯化后的目的蛋白作为抗原免疫家兔制备多克隆抗体。(7)ELISA检测兔抗XBP1u血清的效价。(8)Western blot检测兔抗XBP1u血清的特异性。研究结果(1)扩增出了目的基因片段XBP1u,经测序XBP1u由785bp组成,为编码261个氨基酸残基的多肽。(2)成功构建了原核表达载体pET32a-XBP1u,并转化入大肠杆菌E.coli BL2l。(3)经IPTG诱导大量表达后,SDS-PAGE电泳显示目的蛋白相对分子量为33KD,主要以包涵体形式表达,经NI2+-NTA柱纯化后,纯度达90%以上,BCA法测得蛋白质含量为1.42mg/ml。(4)Western blot对目的蛋白进行分析,由于重组载体pET32a-XBP1u中的pET32a带有His标签,因此可以采用anti-His抗体和anti-XBP1u抗体同时检测XBP1u的抗原性。(5)ELISA检测兔抗XBP1u血清效价为1:64000。(6)免疫印迹检测兔抗XBP1u血清的特异性。结论成功构建pET32a-XBP1u原核表达质粒,转化到大肠杆菌E.coli BL2l表达菌获得了目的蛋白XBP1u,表达量较高,纯度较高;并进一步制备了纯度较高的抗XBP1u多克隆抗体,为研究XBP1u的生物学功能及其他相关后续研究奠定基础。更多还原

【Abstract】 ObjectivesIn eukaryocyte, X-box binding protein 1 (XBP1) has many biological function as a transcriptional activator, it has wide biology effect, participate in many physiological and pathological procession. There has been many focus on the influence of XBP1 on diseases. XBP1 has two forms and XBP1u is its unspliced style, which has transcriptional activity, too. In this study, we make use of molecular biologic technology just as the polymerase chain reaction (PCR)、gene cloning and so on, to construct the prokaryotic expression plasmid pET32a-XBP1u. The recombinant plasmid was induced expression in E.coli BL2l, then we got lots of XBP1u protein with cytoryctes, expression. Furthermore, the purified XBP1u protein was generated by using Ni-NTA affinity chromatography, and we use Western blot to analysis our interest protein. The successful production of purified and exact XBP1u protein was used to immunize the rabbits, then the polyclonal antibody was gained. This will be a basis of further studying of structure and biological function of XBP1u . Methods(1) Abstraction of total RNA from hepatoma carcinoma cell (HepG2) and two steps , RT-PCR was used to amplify the target gene XBP1u.(2) XBP1u was inserted into the pGEM-T vector by using molecular cloning technology to construct plasmid pGEM-T -XBP1u.(3) Construct the prokaryotic expression plasmid pET32a-XBP1u by using molecular cloning technology.(4) The prokaryotic expression plasmid pET32a-XBP1u was induced by IPTG in E.coli BL2l, we got the interest protein. The interest protein was analyzed by SDS-PAGE and purified by Ni2+-NAT chromatography. Its protein concentration was detected by BCA methods.(5) We use Western blot to analysis whether our interest protein was exact.(6) The target protein was used to immunize the rabbits.(7) The titer of the antiserum was detected by ELISA.(8) The specificity of the rabbits, antiserum was detected with Western blot.Results(1) The target gene XBP1u was amplified, and the interest gene was sequenced as 785bp, the interest protein encoded polypeptides of 261 amino acid residues.(2) The prokaryotic expression plasmid XBP1u has been constructed successfully, and transformed it into E.coli BL2l.(3) The expression of pET32a-XBP1u was induced by IPTG, SDS-PAGE showed that the interest fusion protein of XBP1u mainly expressed as inclusion bodies. After purified, the purity of target protein was over 90% and its concentration was 1.42mg/ml.(4) Western blot to analysis the antigenicity of interest protein, and the interest protein can do immune reaction with the His tag antibody. Due to pET32a of pET32a-XBP1u has His tag on it, we can use anti-His antibody and anti-XBP1antibody to detect our interest protein, then we can conclude that this interest protein was XBP1u.(5) The titer of the antiserum was 1:64000.(6) The specified band was found in Western blot.ConclusionsThe prokaryotic expression plasmid XBP1u has been constructed successfully, and transformed it into E.coli BL2l, then we got lots of XBP1u protein, it has higher purity. We gained the polyclonal antibody of XBP1u. This will be a basis of further studying of structure and biological function of XBP1u.更多还原