人hBD2高表达易分离逆转录表达载体构建  

Construction for retroviral expression vector of high expression and easy isolation of human hBD2

作　　者：杜剑平[1] 陈正堂[1] 张青[1] 

机构地区：[1]第三军医大学新桥医院全军肿瘤中心,重庆400037

出　　处：《重庆医学》2004年第11期1681-1683,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目 (30 1 70 4 0 4 )

摘　　要：目的　通过现代分子生物学技术 ,构建人pLNCX2 CEA信号肽 EK激酶 hBD2抗菌肽逆转录表达载体 ,为下一步在真核细胞中高表达hBD2奠定基础。方法　PCR法将CEA信号肽、EK激酶识别位点序列、上下游酶切位点及保护性碱基克隆在一个片段上 ,酶切后 ,T4DNA连接酶将此片段定向连接在含hBD2基因片段的 pBluescriptSK质粒上 ,经过反复构建 ,最终完成pLNCX2 CEA EK hBD2 (成熟肽 )融合基因及其载体 ,并采用逆转录病毒法将融合基因整合到宿主细胞中。 结果　构建到逆转录表达载体pLNCX2上的目的基因双酶切后琼脂糖电泳及测序正确 ,RT PCR检测出宿主细胞融合基因表达。结论　成功构建pLNCX2 CEA EK hBD2逆转录表达载体 ,并整合入宿主细胞中表达。Objective To construct retroviral expression vector of high expression and easy isolation of human hBD2.Methods With designed proper primer, PCR amplifies fusion gene sequence:CEA signal peptide-EK sequence then it is inserted upstream of hBD2 in MCS of pBluescript SK-hBD2 by T4 ligase, with the similar method,thus retroviral expression vector expressing pLNCX2-CEA-EK-hBD2(mature peptide) was constructed. With help of PT67 cell, the CEA-EK-hBD2(mature peptide) is integrated in A549 cell. Results The RT-PCR shows that the A549 cell transcript CEA-EK-hBD2(mature peptide).Conclusion Retroviral expression vector of high expression and easy isolation of human hBD2 was constructed in A549 cell successfully.

关 键 词：CEA信号肽 HBD-2 融合基因 克隆载体 逆转录病毒表达载体 

分 类 号：R346[医药卫生—基础医学]