肿瘤坏死因子相关的凋亡诱导配体（TRAIL）基因的改造及其在毕赤氏酵母菌中的表达分析

发布时间：2018-01-02 18:33

本文关键词：肿瘤坏死因子相关的凋亡诱导配体（TRAIL）基因的改造及其在毕赤氏酵母菌中的表达分析　出处：《华南热带农业大学》2005年硕士论文　论文类型：学位论文

【摘要】：肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis inducing ligand, TRAIL)是肿瘤坏死因子(tumor necrosis factor, TNF)基因超家族众多成员之一,能通过死亡受体的参与而诱导细胞凋亡。 TRAIL与肿瘤细胞膜表面特殊的复杂的受体系统(两个死亡受体和三个诱骗受体)相互作用。由于它的三聚体形式能诱导许多转化细胞凋亡却不会杀伤正常细胞的特性而有望成为极有潜力的治疗肿瘤的药物,引起研究者极大的兴趣。本研究利用甲醇毕赤氏酵母表达系统对TRAIL基因进行了表达研究和高效表达菌株的筛选。以pGEM-TRAIL为模板,设计了三对引物P1、P2、P3、P4、P5、P6,利用套叠PCR技术成功地获得了含有KEX2位点和ATTTA位点以及同时含有这两个突变位点的可溶性TRAIL基因片段,并成功地克隆到E.coli DH5α中扩增。将改造后的TRAIL基因连接到甲醇毕赤酵母Pichia pastoris的YIP型表达载体上,通过Zeocin抗生素筛选,Xba Ⅰ/Xho Ⅰ双酶切鉴定,获得重组表达载体pPICZ_α-A-TRAIL_(unmodified)、pPICZ_α-A-TRAIL_(KEX2 mutation)、pPICZ_α-A-TRAIL_(ATTTA mutation)、pPICZ_α-A-TRAIL_(KA double mutation)。利用电转化技术将重组表达载体转化毕赤酵母GS115,经Zeocin抗生素筛选、PCR检测获得阳性的酵母工程菌株pPICZ_α-A-TRAIL_(unmodified)/GS115、pPICZ_α-A-TRAIL_(KEX2 mutation)/GS115、pPICZ_α-A-TRAIL_(ATTTA mutation)/GS115、pPICZ_α-A-TRAIL_(KA double mutation)/GS115。摇瓶培养确定毕赤酵母工程菌摇瓶最适pH值为5.5-6.5;最佳甲醇诱导浓度为1%;最适诱导周期为96 h。利用SDS-PAGE进行高表达菌株的筛选,得到高表达菌株,表达水平分别达到71.5 mg/L(对照)、127.5 mg/L、114 mg/L和183 mg/L,这说明改造后的菌株表达量比未突变的菌株表达量有较明显的提高。利用CM-柱层析对重组蛋白进行了初步纯化研究。用Western blot检测表明重组蛋白具有TRAIL抗原活性。MTT法检测表达菌株发酵产物显示重组蛋白具有生物活性。
[Abstract]:TNF - related apoptosis inducing ligand ( TRAIL ) associated with tumor necrosis factor is one of the many members of tumor necrosis factor ( TNF ) gene superfamily , which can induce apoptosis by the involvement of death receptor . TRAIL interacts with a specific complex receptor system ( two death receptors and three decoy receptors ) on the surface of the tumor cell membrane . Because of its trimer form , it is expected that many transformed cells will not kill normal cells but are expected to be extremely potential drugs for the treatment of tumors , causing great interest in the researchers . Three pairs of primers P1 , P2 , P3 , P4 , P5 and P6 were designed by using Pichia pastoris expression system . Three pairs of primers P1 , P2 , P3 , P4 , P5 and P6 were designed . The modified TRAIL gene was ligated to YIP expression vector of Pichia pastoris , and was screened by Zeocin antibiotic and Xba I / Xho I double digestion . The recombinant expression vector pZ伪 - A - TRAIL _ ( inducer ) , ZZ _ 伪 - A - TRAIL _ ( ATTTA mutation ) and ZZ _ 伪 - A - TRAIL _ ( KA double mutation ) were obtained . The recombinant expression vector was transformed into Pichia pastoris GS115 by electroporation . After Zeocin antibiotic screening , the positive yeast engineering strains were screened by PCR . The positive yeast engineering strains were identified by PCR . The positive yeast strains were GS115 , ZZ _ 伪 - A - TRAIL _ ( KEX2 mutation ) / GS115 , ZZ _ 伪 - A - TRAIL _ ( ATTTA mutation ) / GS115 , ZZ _ 伪 - A - TRAIL _ ( KA double mutation ) / GS115 . pPICZ_伪-A-TRAIL_(KEX2 mutation)/GS115,pPICZ_伪-A-T High - expression strains were screened by SDS - PAGE . The expression levels of the strains were 71.5 mg / L ( control ) , 127.5 mg / L , 114 mg / L and 183 mg / L , respectively . The recombinant protein was purified by CM - column chromatography . Western blot was used to detect the activity of recombinant protein .

【学位授予单位】：华南热带农业大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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