人HER2／neu胞外区在毕赤酵母中的表达及鉴定

Expression and identification of recombinant human HER2/neu extracellular domain in Pichia pastoris

目的在毕赤酵母中高效表达HER2/neu胞外区,制备具有与天然HER2/neu相同结构和生物学活性的重组人HER2/neu胞外区(rhHER2/neu ECD)。方法用RT-PCR法自人mRNA中克隆her2/neu ECD基因,构建真核表达载体pPICZα/her2/neu ECD。经核酸测序确证后,电转化毕赤酵母菌株X-33。用PCR法筛选基因转染后Zeocin抗性阳性的克隆,用SDS-PAGE法筛选高表达HER2/neu ECD工程菌,用Western印迹法鉴定甲醇诱导的培养上清液中的HER2/neu ECD。结果经RT-PCR法克隆的her2/neu ECD基因序列与GenBank登录的cDNA序列一致。将her2/neu ECD基因定向插入pPICZα,构建pPICZα/her2/neu ECD真核表达载体,经电转化获得Zeocin抗性阳性的克隆。SDS-PAGE和Western印迹法分析证实了甲醇诱导的培养基上清中含有HER2/neu ECD,分子量110kDa。HER2/neu ECD表达量达120mg/L。结论克隆her2/neu ECD基因并构建和筛选出高效表达HER2/neu ECD的毕赤酵母工程菌。

Objective To study efficient expression of Homo sapiens HER2/neu extracellular domain （ECD） in Pichia pastoris, and to offer a technical prerequisite for the further study of HER2/neu ECD as a vaccine in tumor immunotherapy. Methods Her2/neu ECD DNA obtained from human RNA by RT-PCR was cloned into vector pPICZα. After the sequence was confirmed , the recombinant vector was transformed into Pichia pastoris via electroporation. Pichia pastoris secreting HER2/neu ECD in culture supernatant were obtained to extract genomic DNA of transformed X-33 and perform PCR and clone primers. SDS-PAGE and Western blot were used to screen high expressed HER2/neu ECD engineering bacteria and to identify HER2/neu ECD in culture supernatant induced by methanol. Results The sequence of her2/neu ECD DNA obtained by RT-PCR cloning was identical with that published on GeneBank. Recombinant expression vector was constructed to obtain positive Zeocin resistance clone by electrotransformation. SDS-PAGE and Western blot analysis showed HER2/neu ECD in culture supernatant induced by methanol, up to 120 mg/L Pichia pastoris effectively and stably expressed HER2/neu ECD, Conclusions Pichia pastoris engineering bacteria effectively expressing HER2/neu ECD could be constructed and screened by cloning her2/neu ECD.