重组羧肽酶原B甲醇酵母构建与分泌表达.docVIP

重组羧肽酶原B甲醇酵母构建与分泌表达 　　摘要：目的构建重组羧肽酶原B甲醇酵母工程菌株。方法将羧肽酶原B编码基因整合入酵母质粒，电转化至甲醇酵母中，His缺陷培养基和G418抗性筛选获得阳性克隆株。用大肠杆菌表达的羧肽酶原B免疫家兔制备羧肽酶原B兔抗血清；经发酵、甲醇诱导后，蛋白质印迹（Western-blotting）免疫检测蛋白质的表达。结果获得高抗体滴度的羧肽酶原B兔抗血清，蛋白质印迹免疫检测重组酵母经甲醇诱导表达后的上清阳性。结论获得了重组羧肽酶原B甲醇酵母工程菌株，可分泌表达重组羧肽酶原B。 　　关键词：羧肽酶原B；甲醇酵母；表达；多克隆抗体制备；蛋白质印迹 　　中图分类号：Q556文献标识码：A文章编号：1672-979X(2008)11-0005-03 　　 　　Construction and Secreted Expression of Recombinant Procarboxypeptidase B in Pichia pastoris 　　LI Su-xia, YUAN Qin-sheng 　　(State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China) 　　Abstract：Objective To construct the recombinant procarboxypeptidase B in Pichia pastoris. MethodsThe yeast plasmid containing procarboxypeptidase B coding gene was constructed, and then transferred to Pichia pastoris. The positive clone was obtained by G418 resistance selection in His- medium. The rabbit anti-rat recombinant procarboxypeptidase B serum was prepared with recombinant procarboxypeptidase B expressed in E. coli. Western-blotting analysis was used to determine the expressed protein with the prepared antiserum after fermentation and methanol induction of recombinant yeast. Results The rabbit anti-rat recombinant procarboxypeptidase B serum with higher antibody titer was got. The result of Western-blotting analysis showed that the supernatant was positive with the antiserum after methanol induction of recombinant yeast. ConclusionThe recombinant procarboxypeptidase B in Pichia pastoris can be constructed with the successful secretion of recombinant procarboxypeptidase B. 　　Key words：procarboxypeptidase B; Pichia pastoris; expression; polyclonal antibody preparation; Western-blotting 　　 　　羧肽酶B（carboxypeptidase B，CPB，EC 3.4.17.2）是一种含锌的胰外肽酶，特异性水解肽链C端的碱性氨基酸：精氨酸、赖氨酸或鸟氨酸，CPB含307个氨基酸，Mr约35×103。天然CPB由前羧肽酶原B（preprocarboxypeptidase B）产生，其N端包含108个氨基酸（其中13个氨基酸为信号肽序列，95个氨基酸组成活性肽部分）片段，并与C端的CPB相连，前羧肽酶原B在转运到内质网的过程中，信号肽被切掉，得不具有酶活性的羧肽酶原B（procarboxypeptiedase B，pCPB）从细胞中分泌，然后通过胰蛋白酶将其酶