Guo Xue-Yan, Shi Yong-Quan, Zhai Hui-Hong, Sun Li, Liu Li-Li, Han Shuang, Lei Ting, Fan Dai-Ming
National Key Laboratory of Cancer Biology, Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Apr;23(4):363-6.
To construct prokaryotic expression plasmid of RPS13, express and purify the protein for the preparation of polyclonal antibody.
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.coli BL21 and screened by ampiline, and then the E.coli 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.
The expression plasmid pET-28a(+)-RPS13 was constructed. The 19 kDa recombined protein was successfully expressed in the E.coli 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.
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.
构建核糖体蛋白S13（RPS13）原核表达质粒，表达并纯化该蛋白以制备多克隆抗体。
采用逆转录聚合酶链反应（RT-PCR）从胃癌多药耐药细胞SGC7901/VCR高表达细胞中扩增RPS13基因。测序后，将该基因克隆至表达载体pET-28a(+)中构建RPS13表达质粒pET-28a(+)-RPS13。将表达质粒转化至大肠杆菌BL21中，用氨苄青霉素筛选，然后用异丙基-β-D-硫代半乳糖苷（IPTG）诱导含表达质粒的大肠杆菌BL21，并用组氨酸标签柱纯化蛋白。用免疫原His-RPS13免疫BALB/c小鼠。通过酶联免疫吸附测定（ELISA）检测效价，获得多克隆抗体。
构建了表达质粒pET-28a(+)-RPS13。19 kDa重组蛋白经IPTG诱导3小时后在大肠杆菌BL21中成功表达，用组氨酸标签柱纯化，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）鉴定。用RPS13蛋白免疫小鼠获得了多克隆抗His-RPS13抗体。基于Western blot，该抗体可特异性识别RPS13。
RPS13已成功表达和纯化，并制备了多克隆抗His-RPS13抗体。本研究可用于制备单克隆抗His-RPS13抗体以及进一步研究RPS13蛋白在肿瘤中的功能。
