正反义人PIN1基因克隆及真核表达载体的构建_熊文化.pdf

20 06 3 0 3 ·217 · PIN1 (4 30 03 0　)　　熊文化　陈安民　郭风劲　张衣北　黄　涛 　　【】　　、PIN 1 (hPIN 1) hPIN 1 、。　 RT-PCR MG-63 RNA 990bp hPIN 1 cDNA , 、 BamH Ⅰ Hind Ⅲ、hPIN 1 , BamH Ⅰ Hind Ⅲ pIRES2-EGFP , 、hPIN 1 phPIN 1。　cDNA ;BamH ⅠHindⅢ, 、5.3 +0.99kb , 。　、 PIN 1 hPIN 1 、。 【】　PIN 1　　　 　　Cloning and constructionof sense and antisenseeukaryotic expressionvector of human PIN1.X iong Wenhua, Chen Anmin, GuoFengj ing et al .Dep artment of Orthop edic, Tongj i Hosp ital, Tongj i Medical College, H uazhong Uni ersity of S ci - ence and Technology, Wuhan 4 30 03 0 【Abstract】　Objective　To clone and const r ct e karyotic expressing vectors of sense and antisense h man PIN 1 (hPIN 1) genes.Methods　Total RNA was extracted from MG-63 cells, then the hPIN 1 cDNA w as amplified by RT-PCR.At the same time the sense and antisense hPIN 1 genes w ere formed by binding BamH Ⅰand Hind Ⅲ in cis and trans-directions.At the end they were cloned into the e karyotic expressing vector pIRES2-EGFP in cis and trans directions sing DNA recombinant tech- nology.The recombinant vectors w ere f rther identified by digestion of BamH Ⅰ and Hind Ⅲ.Results　Seq encing analysis re- vealed that the orientation of the ligations and the reading f rame w ere correct.After digested by BamH Ⅰ and Hind Ⅲ, two fragments of 5.3 kb and 0.898 kb respectively w ere formed in sense and antisense e karyotic expressing vectors.Elect ro- phoretic res lts w ere completely coincident with theoretical calc lation.Conclusion　H man PIN 1 sense and antisense genes were s ccessf lly cloned and e karyotic expressing vectors w ere s ccessf lly constr cted. 【 ey words】　PIN1 　Isomerase　Antisense gene　Ee karyotic expressing vector 　　PIN 2 0 Invitrogen ; ,, cy- BamH ⅠHind ⅢNew England Bio- [1] clinD1、β-catenin、p53 ; labs ; DNA Roche 、、、PIN, ;X-Gal IPTG [2] 。PIN Omega , 。