人脑胶质瘤特异抗原肽白介素13α2受体的原核表达

Prokaryotic Expression and Purification of IL-13Ra2 Antigen in Human Gliomas

目的:构建白介素13 α2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pMDl9 Simple T载体连接转入DH5α菌克隆,筛选重组阳性菌落,用Xhol和HindⅢ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-13R α2抗原肽,并利用镍柱进行纯化回收,SDS-PACE检测表达蛋白。结果:成功扩增出IL-13Rα2基因序列,大小为1142 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。

Objective： To construct the expression plasmid of the Interleukin-13α2 Receptor （IL-13Rα2） and to detect in vitro prokaryotic 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 T vector was connected and transfered to DH5 α. Positive colonies were screened and recombinated. The IL-13Rα2 was digested from recombinant cloning plasmid using Xho I and Hind III, and was connected to the expression plasmid pET-28a that was also digested by the same enzymes. It was then transfered to the BL21（DE3）. After the recombinant plasmid was abstracted for digestion and sequencing, the prokaryotic expression of IL-13Rα2 antigenic peptide was conducted by the induction of IPTG. Nickel column was used for recovery and purification, and SDS-PAGE was conducted to detect the protein expression. Results： The IL-13Rα2 antigen was successfully expressed and the molecular weight was 60kD, which existed with the modality of inclusion bodies. Conclusion： The object gene IL-13Rα2 could be obtained from U251 glioma cell line by RT-PCR and connected to the expression plasmid pET-28a very successfully. The expression plasmid of IL-13Rα2 gene can be successfully constructed in vitro and can be successfully expressed and purified by IPTG induction to obtain the IL-13Rα2 antigenic peptide.