联合调控CGRP和PPARγ基因的重组载体构建与鉴定  

Construction and idendification of recombinant vectors for combined regulating CGRP and PPARγ gene

作　　者：李岱峰[1] 李劲峰[1] 孙俊魁[1] 王义生[1] 

机构地区：[1]郑州大学第一附属医院骨科河南省高等学校临床医学重点学科开放实验室,450052

出　　处：《中国实用医刊》2016年第20期1-4,共4页Chinese Journal of Practical Medicine

基　　金：国家自然科学基金资助项目（81572216）

摘　　要：目的构建与鉴定联合调控降钙素基因相关肽（CGRP）和过氧化物酶体增殖子活化受体-γ（PPARγ）基因的重组载体。方法将CGRP基因完整ORF克隆，设计其两端带MluI酶切位点，与TGFP融合蛋白表达单元间用一段柔性链连接，CGRP基因ORFcDNA纯化后与线性化pGFP-V-RS载体连接；按照siRNA的设计原则，设计靶向PPARγ的siRNA寡核苷酸靶序列，将发卡样siRNA寡核苷酸靶序列退火后克隆入线性化pGFP-V-RS载体；再在此载体中克隆入CGRPORFcDNA片段。进而将此3个质粒做酶切及测序鉴定。结果表达CGRP基因的pG-FP-V-RS-exCGRP、沉默PPARγ基因表达的pGFP-V—RS-siPPARγ和表达cGfuP且同时沉默PPARγ的pGFP—V-RS-siPPARγ-exCGRP，经酶切及测序鉴定的结果与设计均完全一致。结论成功构建出联合调控CGRP表达和沉默PPARY的双基因重组载体。Objective To construct and identify the recombinant vectors for combined regulating calcitonin gene-related peptide （CGRP） and peroxisome proliferator-activated receptor γ（PPARγ）. Methods Clone the complete CGRP ORF gene, the design of its ends with MluI restriction sites, fusion protein between the connecting unit and TGFP some flexible chain, CGRP gene ORF cDNA after purification connected with the linearized pGFP-V-RS Vector, in accordance with the design principles of siRNA, design of siRNA oligonucleotides targeting PPARγ target sequence, the hairpin siRNA oligonucleotide target sequence annealed and cloned into the linearized pGFP-V-RS vector; then cloned into this vector CGRP ORF cDNA fragment. And then do this three plasmid restriction analysis and sequencing. Results Expression of CGRP gene pGFP-V-RS-CGRP obtained, pGFP-V-RS-siPPARγ and silencing the expression of PPARγ gene expression of CGRP and simultaneously silence PPARγ pGFP-V-RS- siPPARγ-exCGRP, by restriction analysis and sequencing results were fully consistent with the design. Conclusions Recombinant vector for combined regulation of expressing CGRP and silencing PPARγ double is successfully constructed.

关 键 词：降钙素基因相关肽 过氧化物酶体增殖子活化受体-γ 载体 联合调控 

分 类 号：R681.8[医药卫生—骨科学]