摘要
提取SD大鼠肝组织总RNA,通过RT-PCR扩增TIMP-1 cDNA片段.将扩增产物克隆至pUCm-T载体上,PCR、限制性酶切及测序证实后,EcoRI和NotI双酶切pUCm-T-TIMP-1重组体,回收TIMP-1,再将其亚克隆到酵母表达载体pPIC9K中,并将阳性克隆进行PCR、酶切和测序分析,构建pPIC9K-TIMP-1表达载体,电击转化到毕赤酵母,通过表型筛选和诱导表达得到蛋白表达工程菌,为进一步研究TIMP-1功能和作用机理等的研究创造了条件.
The fragment of TIMP-1 cDNA has been amplified from total RNA extracted from SD rat liver tissues by reverse transcription and polymerase chain reaction and has been cloned into pUCm-T vector to construct the clone plasmid pUCm-T-TIMP-1. After identification by PCR, restriction enzyme digestion and nucleotide sequencing, the cloned gene are digested by restrictive endonuclease digestion （EcoR Ⅰ and Not Ⅰ） and subcloned into the eukaryotic expression vector pPIC9K of Pichia pastoris to construct a recombinant vector pPIC9K-TIMP-1 which is confirmed by PCR, restriction endonuclease and digestion and nucleotide sequencing. Then the vector is transformed into Pichia pastoris by electroporation, through phenotype selection and induced with methanol the expression cell is obtained. The experiment provides foundation to further study the mechanism of T1MP-1＇s structure and function.
出处
《河南师范大学学报（自然科学版）》
CAS
CSCD
北大核心
2008年第4期124-127,共4页
Journal of Henan Normal University(Natural Science Edition)
基金
河南省科技攻关项目(0624240008)
河南省动物重点学科资助
