摘要
目的:克隆并在毕赤酵母中表达小鼠sIL-13Rα2基因。方法:从小鼠脾脏细胞中提取总RNA,逆转录为cDNA,应用分段PCR的方法将sIL-13Rα2基因定向克隆至酵母表达质粒pPICZα-A,并转染至毕赤酵母菌表达系统进行目的蛋白表达。目的蛋白行SDS-PAGE和Western分析。结果:克隆出可溶性IL-13Rα2目的基因片段,构建重组pPICZα-A/sIL-13 Rα2表达载体,成功转染至毕氏酵母菌,诱导表达出可溶性IL-13Rα2目的蛋白。结论:已成功克隆并在毕赤酵母中表达了小鼠可溶性IL-13Rα2目的基因,为应用sIL-13Rα2蛋白治疗哮喘及其他过敏性疾病奠定了基础。
Objective：To clone and express routine soluble interleukin 13 receptor α2（sIL-13Rα2）gene in Pichia Yeastpastoris. Methods：The total RNA extracted from spleen cells of BALB/c mouse as template, sIL-13Rα2 cDNA enconding extracellular domain of sIL-13Rα2 chain was obtained by RT-PCR. Using eDNA template the cloned target gene was amplified by PCR. Target gene was digested with restriction enzyme and cloned into Pichia Yeast expressing vector. Recombinant plasmids was transfected into Pichia Yeast pastoris for expressing target protein, and the target protein was identificated by SDS-PAGE and Western blot. Results： sIL-13Rα2 target gene was successfully cloned into the recombinant pPICZα-A/sIL-13Rα2 expression vector. The recombinant expressing vector was successfully transfected into Pichia Yeast pastoris, and sIL-13Ra2 protein was positively expressed by identification. Conclusion： Murine sIL-13Rα2 target gene is obtained and reeombinantly expressed into the Pichia Yeast pastoris vector. It may establish the foundation for further application of sIL-13Rα2 in treatment asthma and other allergic diseases.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2009年第6期483-486,共4页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(30770926)
关键词
sIL-13Rα2基因
克隆
表达
毕赤酵母菌
Soluble interleukin 13 receptor α2 gene
Clone
Expression
Pichia Yeast pastoris
