蚯蚓纤溶酶7#组分基因在甲醇酵母中的克隆与表达  

Gene Cloning and Expression of Component 7# of Earthworm Fibrinolytic Enzyme in Pichia pastoris

作　　者：赵晓瑜[1] 张冬[1] 李亚东[1] 

机构地区：[1]河北大学生命科学学院,河北保定071002

出　　处：《河北大学学报（自然科学版）》2005年第6期633-638,共6页Journal of Hebei University(Natural Science Edition)

基　　金：河北大学自然科学基金资助项目

摘　　要：为了使蚯蚓纤溶酶(EFE)最佳活性组分EFE_7#基因在甲醇酵母(Pichiapastoris)中获得表达,构建了表达载体pPIC9K_EFE,通过对电击条件的优化,转化率达到5×103个/μgDNA.为了便于筛选阳性克隆,以该基因的原核表达产物为抗原免疫动物,获得了与天然EFE_7#组分具有相同免疫特异性的抗体.用该抗体为探针对上述转化子进行筛选,得到了上百株阳性重组子.RT_PCR和westernblotting证明,EFE_7#基因在这些重组子中获得了表达,表达产物相对分子质量高于天然产物.To express gene of the component 7 # of earthworm fibrinolytic enzyme （EFE） in Pichia pastoris, the vector pPIC9K-EFE-7# was constructed. Using eletro-transformation a lot of transformants of pPIC9K-EFE （5×10^3个/μg DNA） were obtained. Antibodies which was immuno-identified with native component 7 # were produced by immunization using products expressed in E. coli. The antibodies against component 7# had been used as probe to screen the positive transformants. RT-PCR and Western Blotting showed the gene of component 7 # was expressed in Pichia pastoris, The molecular weight of products expressed were higher than that of the native one.

关 键 词：蚯蚓纤溶酶 甲醇酵母 表达 抗体 筛选 

分 类 号：Q786[生物学—分子生物学]