反义bcl-2基因逆转录病毒表达载体的构建

Construction of Bcl-2 Antisense RNA Expressed by Defect Retroviral Vector

目的 克隆 bcl- 2基因 ,构建其不同反义核酸的逆转录病毒表达载体。 方法 设计一对两端带有Eco R 酶切位点的引物 ,RT- PCR克隆含 bcl- 2全部编码区的 848bp片段 ,并连接到 T载体 ,测序正确后正反向亚克隆到 p L XSN中 Eco R 位点 ;从 p Bluescript SK(- ) bcl- 2中用 Bam H 切下含有 bcl- 2阅读框起始部位的部分编码区亚克隆到 p L XSN Bam H 位点。 结果 构建带有正反向含 bcl- 2阅读框的全部编码区和部分编码区的逆转录病毒表达载体。 结论 在已知基因序列克隆中 ,联合 RT- PCR和 T载体是一个简单、快速、有效的方法 。

Objective\ To clone bcl\|2 gene and develop two different bcl\|2 antisense RNA expressed by defect retroviral vector.\ Methods\ Both upper and down primers with the end of EcoRⅠ site were designed to amplify 848 bp cDNA fragement including the whole bcl\|2 exon by RT\|PCR.\ After confirmation of the sequences of cloned bcl\|2 exon by T vector, both forward and backward of bcl\|2 cDNA were subcloned into the EcoRⅠ site of retroviral vector pLXSN.\ In the other way, 600 bp cDNA fragement with the beginning of coding bcl\|2 exon was obtained by digestion of pBluescriptⅡ SK(-) bcl\|2 with BamHⅠ restriction enzyme and also subcloned into the BamHⅠ site of retroviral vector pLXSN.\ Results\ Retroviral vectors with both forward and backward, whole and part of bcl\|2 exon were successfully constructed.\ Conclusion\ Under the knowledge of targeting cDNA sequences, the approach for clonning of targeting gene by combination of RT\|PCR with T vector is simple, rapid, effective, and offering the underlying bases of gene transfer and expression.