人血清白蛋白基因的克隆及其真核表达载体构建

Gene cloning of human serum albumin and the construction of eukaryotic expression vector

利用RT-PCR扩增并克隆了含人血清白蛋白序列长约1.8 kb的片段,酶切鉴定并进行序列分析,测序结果与GenBank中登录的人血清白蛋白cDNA序列的同源性为97%;以鹌鹑卵清蛋白5’调控区POV来启动HAS的表达。用限制性内切酶kpnⅠ和HindⅢ双酶切质粒pOV和pHSA,然后用T4连接酶定向连接成重组质粒pOV-pHSA,经酶切鉴定,两者成功融合。

About 1.8 kb sequence of the fragment of human serum albumin containing were successfully amplified by RT-PCR.The purpose of the initia gene were identified with the help of electrophoresis. The DNA fragment was cloned into vector pGEM-T and transformed into E.coli DH5a host bacteria. Xba Iendonuclease digestion and sequencingfrom selected positive cloned bacteria. The fragment was sequenced,and it was compared with serum albumin cDNA gene of humanin GenBank.The results indicated the homology were 97%.Using 5’-flanking of ovalbumin in quail for the control sequence to activate the expression of HAS.With restrictive interior contact enzyme kpnI with HindⅢ cuts material particle pOV and pHSA,then connects reorganization material particle for pOV-pHSA with the T4 ligase. The fusion vector was sequenced by enzyme digestion.