人源化卵巢癌单链抗体基因的构建与表达  

Construction and expression of recombinant ovarian cancer gene ScFv in pichia pastoris

作　　者：许天敏[1] 崔满华[1] 谷丽萍[1] 王丁丁[2] 苏曼曼[2] 王文加[2] 张赢予[2] 焦平[2] 

机构地区：[1]吉林大学第二医院妇产科,吉林长春130041 [2]吉林大学再生医学研究所生物工程教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2008年第2期217-220,共4页Journal of Jilin University：Medicine Edition

基　　金：吉林省科学技术厅基金资助课题(20050410-1)

摘　　要：目的:通过基因工程技术获得完全人源化的卵巢癌患者自身的单链抗体并在毕氏酵母中获得表达,为人源化单链抗体(ScFv)在卵巢癌的诊断与治疗的应用提供实验基础。方法:用TRIZOL法自卵巢癌患者的淋巴细胞中提取RNA,利用SOEingPCR方法分别获得VH、VL基因,构建真核表达载体pPICZα/ScFv。电转化毕赤酵母菌株X-33。用PCR法筛选转染阳性克隆,SDS-PAGE法鉴定甲醇诱导的培养上清液中的ScFv的表达,筛选高表达工程菌。结果:构建ScFv基因,VH与VL连接后得到700bp左右的条带,ScFv基因在毕赤酵母中获得表达,在26000处出现目的条带。结论:获得ScFv基因及其真核表达载体以及高效表达ScFv的毕赤酵母工程菌。Objective To obtain ScFv gene from ovarian cancer patients with gene recombinant technique and express it into pichia pastoris in order to provide experimental basis of application of ScFv in diagnosis and treatment of ovarian cancer. Methods The total RNA of lymphocytes cell was extracted from ovarian cancer patients by TRIZOL. VH and VL gene were obtained by SOEingPCR. pPICZa/ScFv was contructed and transformed into X-33 cells. The expression was induced by pichia pastoris. The engineer bacteria with high effective expression of ScFv was screened. Results ScFv gene was constructed successfully. ScFv gene was about 700 bp and protein was induced by pichla pastoris and it was identified that its molecular weight was about 26 000. Conclusion ScFv gene, pPICZa/ScFv and pichia pastoris engineer bacteria with high effective expression of ScFv are obtained.

关 键 词：卵巢肿瘤 单链抗体 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]