牛源整合素β_1亚基的克隆及表达载体的构建

Cloning of bovine integrin β_1 gene and construction of its expression vector

参照已公布整合素β1亚基的基因组序列,设计并合成了对克隆引物,以RT-PCR方法从牛肺组织扩增出约2 400 bp的β1基因,经回收纯化连入PGEM-T载体,测序.依照测序结果设计1对原核表达引物,1对真核表达引物,分别以克隆载体PGEMβ1为模板扩增目的片段,经相应的EcoR I、XhoI和EcoR I、XbaI酶切纯化后,在T4DNA连接酶的作用下,定向亚克隆到原核表达载体pET-28a和真核表达载体pCDNA3.1zero(+)中,PCR、酶切鉴定及序列测定表明,成功构建了β1亚基原核表达载体pETβ1及真核表达载体pCDNAβ1.

Two pairs of specific primers referring to the bovine integrin betal gene in Genebank were designed and synthesized,a 2400 bp fragment was produced by reverse transcription polymerase chain reaction （RT--PCR） from bovine lung. The amplified fragment was cloned to pGEM--T easy vector, confirmed by sequenceing. Then a pair of prokaryotic and euokaryotic expression primers was designed on the basis of sequenceing results. The expression sequences from the cloned vector pGEMbetal were amplified and digested respe fied betal gene of ctively by the restricted enzyme EcoR I,Xho I and EcoR I,Xba I. The digested and puribovine were cloned into prokaryotic expression vector pET-28a and euokaryotie expression vector pCDNA3, lzero（＋） . The result showed that the euokaryotic expression recombinant plasmid pCDNAbl and prokaryotic expression recombinant plasmid pETbl were successfully constructed.