抗心肌肥大多肽GCIP表达载体的构建

Construction of expression vector for prophylactic myocardial hypertrophy GCIP polypeptide

目的 化学合成DNA片段后利用基因工程技术 ,克隆入含有T7启动子的原核表达质粒。方法 采用分二段化学合成G蛋白竞争性抑制肽 (G proteincompetedinhibitionofpeptide ,GCIP)基因的方法 ,酶切后先定向克隆到pEGFP N1质粒中 ,再用XhoⅠ和SmaⅠ切下含完整基因的片段 ,插入XhoⅠ和SmaⅠ处理的pIVEX2 3 MCS质粒 ,构建能在EcoliBL2 1(DE3 )pLysE和RTS5 0 0系统中表达的质粒。经转化DH5α大肠杆菌 ,筛选出阳性转化子 ,并用酶切及测序鉴定。结果通过基因工程技术 ,构建了pIVEX2 3MCS GCIP表达质粒 ,经测序鉴定结果与设计的完全相符。

Objective To construct a recombinant plasmid for expressing peptides of G protein competed inhibition of peptide (GCIP) under the control of T7 promoter. Methods Two nucleotide fragments were synthesized, and double stranded oligonucleotide was inserted into pEGFP N1 plasmid vector. The total GCIP gene was digested with Xho Ⅰ and Sma Ⅰ, and subcloned into the plasmid pIVEX2.3 MCS for expression in E. coli BL21(DE3)pLysE or RTS500 system. Positive clones were screened and identified with restriction endonuclease, and pIVEX2.3MCS GCIP plasmid was identified by DNA sequencing. Results By DNA recombination techniques, the pIVEX2.3MCS GCIP plasmid was constructed and confirmed by DNA sequencing. Conclusion The expression plasmid, which includes the GCIP gene is under the control of T7 promoter, has been cloned successfully.