人核糖体蛋白S13的原核表达、纯化及多克隆抗体的制备  

Prokaryotic expression and purification of RPS13 and prepartion of polyclonal antibody against RPS13

作　　者：郭雪艳[1] 时永全[1] 翟惠虹[1] 孙力[1] 刘理礼[1] 韩霜[1] 雷婷[1] 樊代明[1] 

机构地区：[1]第四军医大学肿瘤生物学国家重点实验室西京医院消化病研究所,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2007年第4期363-366,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30530780;30670971);第四军医大学"后备人才"基金(2005年)

摘　　要：目的克隆人核糖体蛋白S13(ribosomal protein S13,RPS13)cDNA全长,构建原核表达质粒,诱导表达、纯化RPS13蛋白并制备其多克隆抗体。方法应用RT-PCR方法从胃癌多药耐药细胞SGC7901/VCR中扩增出RPS13的cDNA全长,利用DNA重组技术构建重组原核表达载体pET-28a(+)-RPS13,双酶切及测序鉴定。将重组载体转化入大肠杆菌BL21中,筛选抗性克隆并经DNA测序证实。IPTG诱导融合蛋白的表达,利用镍离子亲和层析柱提纯融合蛋白,经SDS-PAGE和Western blot鉴定。用纯化的融合蛋白His-RPS13免疫BALB/c小鼠,ELISA法测定抗体效价,饱和硫酸铵沉淀法纯化抗体,Western blot检测抗体活性。结果RT-PCR扩增片段与预计目的基因片段大小一致;双酶切鉴定表明,重组表达载体pET-28a(+)-RPS13构建成功,DNA测序结果显示所获基因序列与GenBank中收录完全相同。IPTG诱导3h后,经SDS-PAGE检测显示在Mr19000处出现一新生条带,与预计的融合白大小一致;利用抗His标签的抗体进行Western blot,结果证实融合蛋白表达成功。其免疫血清经Western blot证实可特异性的识别蛋白RPS13。结论成功地获得了RPS13的编码基因序列,并获得了纯化的RPS13蛋白质,为制备RPS13的单克隆抗体、进一步研究RPS13在肿瘤中的作用奠定了基础。AIM： To construct prokaryotic expression plasmid of RPS13, express and purify the protein for the preparation of polyclonal antibody. METHODS： RPS13 gene was amplified by reverse transcription polymerase chain reaction （RT-PCR） from the highly expressed cell of gastric multidrug cell SGC7901/VCR. After sequenced, the gene was cloned into the expression vector pET-28a（ ＋ ） to construct RPS13 expression plasmid pET-28a（ ＋ ）-RPS13. The expression plasmid was transformed into the E. coil BL21 and screened by ampiline, and then the E. coil BL21 containing the expression plasmid was induced by IPTG and the protein was purifed by Histag column. BALB/c mice were immunized by the immunogen His-RPS13. The titer was measured by ELISA and the polyclonal antibody was. obtained. RESULTS： The expression plasmid pET-28a（ ＋ ）- RPS13 was constructed. The 19 kDa recombined protein was successfully expressed in the E. coil BL21 by IPTG for 3 hours, purified by Histag column and confirmed by SDS-PAGE and Western blot. The polyclonal anti-His-RPS13 antibody was obtained by immunizing the mice with RPS13 protein. RPS13 could be specifically recognized by the antibody based on Western blot. CONCLUSION： RPS13 has bene expressed and purified successfully and polyclonal anti-His-RPS13 antibody has been prepared. Our study can be used for the preparation of monoclonal anti-His-RPS13 antibody and for further research of the function of the RPS13 protein in tumor.

关 键 词：核糖体蛋白S13 原核表达 融合蛋白 纯化 多克隆抗体 

分 类 号：Q786[生物学—分子生物学]