摘要
将猪囊虫磷蛋白P2基因克隆到毕赤酵母表达系统分泌性表达载体pPIC9K中 ,构建了重组表达载体pPIC9K P2。经测序证明基因序列完全正确 ,从而大量制备重组质粒pPIC9K P2 ,并用SalⅠ、BglⅡ两种内切酶分别线性化 ,然后分别电转化毕赤酵母菌种GS115 ,采用G418抗性梯度筛选得到高拷贝重组菌株 ,利用MM和MD培养基鉴定重组菌株Mut表型 ,然后用甲醇进行诱导表达 ;并对表达产物通过SDS PAGE、Western blot和ELISA分析 ,结果表明 ,重组菌株成功地分泌表达了分子量大小为 12 6kD ,表达量占分泌总蛋白的 3 3 % ,且具有免疫反应活性的重组磷蛋白 。
Cysticercosis is caused by the metacestode form of Taenia solium-Cysticercus cellulosae and it causes great economic losses and threatens the people's health. There are some problems on how to control cysticercosis, in order to resolve the key problem that the native antigen to diagnose and prevent cysticercosis is very limited and is not satisfied, Pichia pastoris Expression System was used to express recombinant P2 protein. The interested P2 gene was got by digesting the pGEM-P2 vector using restriction endonuclease,then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E.coli. Positive recombinant plasmids were selected、sequenced and named pPIC9K-P2 and it was linearized by SalⅠand BglⅡ,then the linear DNA transfored into Pichia pastoris GS115 by electroporation. The recombinant expression vector pPIC9K-P2 integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. The transformants were screened for multicopy recombinants using G418 and were distinguished for Mut phenotypes by MD and MM. Two different phenotypes were generated-HIS +MUT +(Methanol utilization plus) and HIS +MUT S (Methanol utilization slow). PCR analysis of the multicopy recombinants indicated that the P2 gene was integrated within the genome of pichia Pastoris.The multicopy recombinants were named GS115/pPIC9K-P2HIS +MUT + and GS115/pPIC9K-P2HIS +MUT S, both HIS +MUT + and HIS +MUT S were induced with methanol. The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of the induced Pichia pastoris contained P2 protein which was accumulated up to 33% of total proteins in the culture supernant and its molecular weight is 12.6kD. The results of the clinical study indicated that the expression P2 protein could be used to diagnose human cysticercosis and swine cysticercosis as diagnosis antigen.
出处
《生物工程学报》
CAS
CSCD
北大核心
2003年第4期424-427,共4页
Chinese Journal of Biotechnology
基金
国家重大基础研究发展规划项目基金资助 (No .G19990 119)~~
关键词
猪囊尾蚴磷蛋白
毕赤酵母
表达
囊虫病
Cysticercus cellulosae, P2 gene, Pichia pastoris, secretory expression, diagnosis antigen
