人卵透明带3基因片断hZ3.3的克隆及其在毕赤酵母中的表达  

CLONING AND EXPRESSION OF THE GENE hZ3.3 ENCODING FRAGMENT OF HUMAN ZONA PELLUCIDA 3 IN PICHIA PASTORS

作　　者：熊波[1] 曹佐武[1] 何柳媚[1] 丰一兴[1] 邱平明[1] 

机构地区：[1]暨南大学生殖免疫所,广东广州510632

出　　处：《南昌大学学报（理科版）》2005年第2期200-204,共5页Journal of Nanchang University(Natural Science)

基　　金：国家自然科学基金资助项目(30271370)

摘　　要：为了探讨透明带与精子的作用机制,人卵透明带3(humanZonaPellucida3,hZP3)第191位氨基酸到319位氨基酸之间的cDNA序列通过PCR方法扩增后,克隆到表达载体pPICZαA上,构建成重组质粒pPICZαA-hZ3.3。该质粒经SacⅠ线性化后电打孔转入Pichiapastoris酵母X-33中,用YPDSZeocin+筛选高拷贝转化子,并用PCR分析鉴定目的基因片断与酵母基因组的整合,阳性转化子用0.5%甲醇诱导,表达目的蛋白。经SDS-PAGE凝胶电泳和Western-blot鉴定分析,结果表明目的基因成功表达,表达产物大小约为37kD。In order to explore the molecular mechanism of zona pellucida-sperm interaction, a DNA fragment (387bp) corresponding to hZP3 peptide fragment 191~319 was amplified by PCR from hZP3 cDNA and then subcloned into the MCS of expression vector pPICZαA. The recombinant plasmid pPICZαA-hZ3.3 was transformed into Pichia pastoris yeast X-33 via electroporation, and then high-copy transformants were screened out on the YPDS plates with high concentration Zoecin. After induction of 0.5% methanol, a specific peptide fragment of 37 kD or so was expressed and secreted out of the cells. Western-blot result showed that the peptide could bind anti-human zona antibody specifically. These suggested a recombinant human zona pellucida fragment hZ3.3 gene expression vector had been successfully constructed and hZ3.3 peptide had been expressed in the engineered yeasts.

关 键 词：人卵透明带3 克隆 PICHIA PASTORIS 表达 

分 类 号：Q786[生物学—分子生物学]