杂交捕获抑制聚腺苷二磷酸核糖聚合酶基因的体外翻译

HYBRIDIZATION ARRESTED TRANSLATION IN VITRO OF POLY(ADP-RIBOSE)POLYMERASE GENE

体外转录载体ｐＳＰ７２—Ｃ１２和ｐＳＰ７２—Ｃ１．４分别经ＸｂａⅠ和ＸｈｏⅠ酶切线性化后，用ＳＰ６ＲＮＡ聚合酶与Ｔ７ＲＮＡ聚合酶进行体外转录得到编码人聚腺苷二磷酸核糖聚合酶基因的ｍＲＮＡ全序列（３．７ｋｂ）及与其５’端编码区的起始密码子ＡＵＧ上游区域互补的反义ＲＮＡ片段（１．４ｋｂ）。等摩尔数的正、反义ＲＮＡ通过酚相乳浊杂交技术进行分子杂交。在网织红细胞裂解物体外翻译系统中，正义ＲＮＡ能正常地翻译出蛋白质；而反义ＲＮＡ与正义ＲＮＡ杂交可阻断翻译的进行。

The full Iength mRNA (4. 0 kb) encoding human poly (ADP-ribose) polymerase and its antisense fragment (1. 4 kb) which is complementary to both coding region and 5' terminus franking region (upstream the initiator codon AUG) have been obtained by transcription in vitro with SP6 RNA polymerase or T7 RNA polymerase from the plasmids, pSP72-C12 and pSP72-C1. 4 ,which were linearized by Xba Ⅰ and Xho Ⅰ,respectively. The sense and antisense RNAs were successfully hybridized through Phenol Emulsion Reassociation Technique. In Reticulocyte Lysate Translation System, mRNA can be translated into protein regularly white its hybridization with antisense RNA may block the translation ,process.