RAGE、NF-κB反义RNA单/双基因共表达载体的构建及序列测定

Construction of Vector of Advanced Glycation Endproducts, NF-κB Single/Double Gene Coexpression and Sequence Analysis

目的 构建晚期糖化终产物受体(RAGE)、核因子κB(NF κB)反义RNA单/双基因共表达载体。 方法 从人脐静脉内皮细胞ECV30 4中提取总RNA ,RT PCR扩增RAGE基因片段;从糖尿病人外周血单核细胞提取总RNA ,RT PCR扩增NF κBp6 5基因片段。分别克隆入PGEM T载体,酶切鉴定阳性克隆,再经PCR反应获得多量的RAGE ,NF κB基因片段,反向插入双启动子真核表达载体pBudCE 4 .1,构建RAGE、NF κB反义RNA单/双基因共表达载体。 结果 RT PCR扩增出RAGE基因片段32 8bp,扩增出NF κBp6 5基因片段36 4bp。测序分析证实目的基因片段正确反向插入3个重组载体。 结论 RAGE、NF κB反义RNA单/双基因共表达载体构建成功。

Objective To construct antisense RNA of receptor of advanced glycation end products(RAGE), NF-κB single/double gene coexpression vector. Methods Human RAGE gene fragment from ECV304 was amplified by RT-PCR, while human NF-κB p65 gene fragment from peripheral blood mononuclear cell of a diabetes patient was amplified by RT-PCR. The two gene fragments were inserted into the PGEM-T vector. The positive clones were identified by restriction end onuclease digestion. RAGE and NF-κB gene fragments from PGEM-T/RAGE and PGEM-T/NF-κB were amplified by PCR. The two gene fragments were oppositely inserted into pBudCE 4.1, which contains two promoter. Three antisense RNA of RAGE, NF-κB single/double gene coexpressive vector were identified by restriction end onuclease digestion and DNA sequence analysis. Results Antisense RAGE and NF-κB gene fragments were amplified and clone into pBudCE 4.1 successfully. Nucleotide sequence analysis showed that the constructed fragments were complemented and opposite to human original gene cDNA. Conclusion Antisense RNA of RAGE、NF-κB single /double gene coexpression vector were successfully construct.