摘要
利用基因工程技术构建表达人Ｃ１抑制物（Ｃ１－ＩＮＨ）的表达质粒ｐＳＶＣ，该质粒与编码二氢叶酸还原酶（ｄｈｆｒ）的表达质粒ｐＳＶ２－ｄｈｆｒ一起共转染ＣＨＯ－ｄｈｆｒ＾－细胞，获得了人Ｃ１－ＩＮＨ在真核细胞中的高效表达，表达产物经ＳＤＳ－ＰＡＧＥ、免疫沉淀及免疫印迹分析，表现为一条分子量在１０６×１０＾２处理的明显反应条带。此外，在９４×１０＾３处尚有一条稍弱的条带。
An expression plasmid pSVC was constructed by inserting the human C1-INH cDNA fragment which was cleaved to remove its 3. untranslated regions into the pSV 2-dhfr vector. This plasmid was co-transfected with a selectable dihydrofolate reductase (DHFR ) gene into DHPR-deficient Chinese hamster ovary cells (CHO-dhfr- ). Efficient expression of human C1-INH was obtained in mammalian cells. The analysis of expression products by SDS-PAGE . Immunoprecipitation and Western-blot showed a clear specific band of 106KD. However ,under this band there was another slightly diffused band of 94KD , which might be a degraded form or an incomplete glycosylated form of C1-INH. The functional activity of expressed products was determined by the inhibition of C1 estrolysis assay. We have also observed the effect of different molar ratio between the target gene plasmid and the selective gene plasmid on the expression level of C1-INH during co-transfection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1996年第4期240-244,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金
关键词
C1抑制物
基因表达
共转染
CHO细胞
C1 inhibitor(C1-INH )
Gene expression
Co-transfection CHO cells
