摘要:
目的:构建白介素13α2受体 (Interleukin-13α2 Receptor, IL-13Rα2) 基因的表达质粒, 并检测其在体外原核表达情况,为脑胶质瘤的免疫治疗奠定基础。方法: 用RT-PCR技术从U251胶质瘤细胞株扩增IL-13Rα2基因, 并与克隆质粒pMD19 Sim?ple T载体连接转入DH5α菌克隆, 筛选重组阳性菌落, 用Xho I和Hind Ⅲ将目的基因从克隆质粒上切下并与同样双酶切后的表达质粒pET-28a连接, 转入BL21 (DE3) 菌, 提取重组质粒进行酶切及测序鉴定正确后, 在IPTG诱导下原核表达IL-13Rα2抗原肽,并利用镍柱进行纯化回收, SDS-PAGE检测表达蛋白。结果: 成功扩增出IL-13Rα2基因序列, 大小为1 142 bp, 并表达纯化出IL-13Rα2抗原肽, 大小为60 kD, 于诱导第3 h表达量最高, 以包涵体形式表达。结论: IL-13Rα2基因表达质粒能在体外成功构建, 并能在IPTG诱导下成功表达及纯化得到IL-13Rα2抗原肽。
Abstract:
Prokaryotic Expression and Purification of IL-13Rα2 Antigen in Human GliomasWenqiao YU1, Zuobing CHEN1, Yun ZHANG1, Zhongyan LIANG1, Shaoyang ZHANG1,Weiming ZHENG2Correspondence to:Weiming ZHENG, E-mail: zhwm61@126.com1Department of Emergency Surgery, The First Affiliated Hospital of Zhejiang University Medical School, Hangzhou 310000, China2Department of Neurosurgery, The First Affiliated Hospital ofWenzhou Medical College,Wenzhou 325000, ChinaAbstract Objective: To construct the expression plasmid of the Interleukin-13α2 Receptor (IL-13Rα2) and to detect in vitroprokaryotic expression of the receptor gene, in order to invetigate the theoretical basis of of immunotherapy for human gliomas.Methods: The object gene IL-13Rα2 was obtained from U251 glioma cell line by RT-PCR, then the cloning plasmid pMD19 Simple Tvector was connected and transfered to DH5α. Positive colonies were screened and recombinated. The IL-13Rα2 was digested fromrecombinant cloning plasmid using Xho I and Hind III, and was connected to the expression plasmid pET-28a that was also digested bythe same enzymes. It was then transfered to the BL21(DE3). After the recombinant plasmid was abstracted for digestion andsequencing, the prokaryotic expression of IL-13Rα2 antigenic peptide was conducted by the induction of IPTG. Nickel column wasused for recovery and purification, and SDS-PAGE was conducted to detect the protein expression. Results: The IL-13Rα2 antigen wassuccessfully expressed and the molecular weight was 60kD, which existed with the modality of inclusion bodies. Conclusion: Theobject gene IL-13Rα2 could be obtained from U251 glioma cell line by RT-PCR and connected to the expression plasmid pET-28a verysuccessfully. The expression plasmid of IL-13Rα2 gene can be successfully constructed in vitro and can be successfully expressed andpurified by IPTG induction to obtain the IL-13Rα2 antigenic peptide.Keywords Gliomas; IL-13Rα2 antigen; Expression plasmid