摘要
目的：用毕赤酵母（Pichia pastoris）表达人透明带hZP3片段,研究其抗生育作用。方法：设计特定引物从hZP3全长序列中PCR扩增794-1282nt基因片段,将扩增片段克隆到酵母表达载体pPICZα上,构建成重组质粒pPICZα-hZP3CT,线性化后的重组质粒导入毕赤酵母中,筛选出高拷贝菌株,甲醇诱导目的蛋白表达。用SDS-PAGE和Western blotting分析表达产物。结果：成功构建酵母表达载体,并在酵母菌株X-33中诱导表达重组蛋白。重组蛋白可以与鼠抗重组人卵透明带hZP3融合肽段的抗血清发生特异结合,表明目的基因成功表达。结论：重组hZP3蛋白已在酵母中成功表达,目的蛋白能与相应的抗体结合。
Aim：In order to investigate the role of the human zona pellucide 3 portein（hZP3） in the fertility regulation,recombinant hZP3 C-terminal peptide was expressed in pichia pastoris yeast. Methods： The hZP3 DNA fragment（794-1282nt） was amplified by PCR from the hZP3 cDNA.The cloned DNA fragment of hZP3 was inserted into an expression vector pPICZα for pichia pastoris.Then,the linearized recombinant plasmid pPICZα-hZP3CT was transformed to Pichia pastoris X-33 by electroporation.The transformant cell carrying high copy hZP3CT gene were selected on the plates with high concentration zeocin.Expression of hZP3 peptide was induced by methanol and verified by SDS-PAGE and Western blotting.Results： The engineered yeast strain containing high copy-gene of the recombinant hZP3 peptide was successfully constructed.Western blotting showed the recombinant protein was expressed after methanol induction.Conclusion：The recombinant hZP3 peptide was successfully expressed in the engineered yeast and reacted specifically with the mouse antiserum against hZP3.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2010年第6期533-538,共6页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金重点项目(21307017)
