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 Simple 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.%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 DH5α.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.
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