重组羧肽酶原B甲醇酵母的构建与分泌表达

Construction and Secreted Expression of Recombinant Procarboxypeptidase B in Pichia pastoris

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。

Objective To construct the recombinant procarboxypeptidase B in Pichia pastoris. Methods The 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. Conclusion The recombinant procarboxypeptidase B in Pichia pastoris can be constructed with the successful secretion of recombinant procarboxypeptidase B.