RNase H在反义寡聚脱氧核苷酸抑制小鼠白血病病毒表达过程中的作用

用与Ｍｏｌｏｎｅｙ小鼠白血病病毒（Ｍｏ－ＭｕＬＶ）５＇端引物序列、ｅｎｖ－ＬＴＲ间序列及ｐｏｌ基因互补的三种反义脱氧寡核苷酸（１４ｍｅｒ，１８ｍｅｒ，３５ｍｅｒ）进行抗小鼠白血病病毒（ＳＲＳＶ）作用研究。发现它们在兔网织红细胞体外翻译系统中能抑制ＳＲＳＶｍＲＮＡ的转译，其中，３５ｍｅｒ作用最强，浓度为８μｍｏｌ／Ｌ时抑制率约４０％１４ｍｅｒ作用较弱，抑制率仅２３％；ＲＮａｓｅＨ能有效降解不对称ＰＣＲ反应合成的单链反义ＤＮＡ（１１９ｍｅｒ）与ＳＲＳＶＲＮＡ杂交体中的ＲＮＡ，加入ＲＮａｓｅＨ后，３５ｍｅｒ和１８ｍｅｒ对ＳＲＳＶｍＲＮＡ转译的抑制率分别由４２％提高到５９％和２７％提高到３３％，但１４ｍｅｒ抑制率无改变。提示反义寡核苷酸能有效地抑制ＳＲＳＶｍＲＮＡ的表达，这种作用呈序列特异性和剂量依赖性。它们可能是通过ＲＮａｓｅＨ对反义ＤＮＡ·ＳＲＳＶｍＲＮＡ杂交体中的ＲＮＡ降解所致。

ROLES OF RNase H IN INHIBITION OF SRS MOUSE LEUKEMIA VIRUS (SRSV) EXPRESSION BY ANTISENSE OLIGONUCLEOTIDES

The oligonucleotides reported here were complementary to the primer region of 5' end (14 mer) , env-LTR gene region (18mer) and pol gene region (35mer) of Mo-MnLV mBNA. They successfully inhibit the expression of SRSV mRNA in the rabbit reticulocyte lysate system. Among these, the 35mer is the most efficiency inhibitor which % inhibition goes to 40% and the 14mer is relatively less efficiency, which % inhibition is only 23%,RNase H could largerly increase the effect of5mer which % inhibition were increased from 42% to 59%, it also increases the effect of 18mer, where as it has made no effect on the 14 mer. Studies also proved this supression of viral expression has sequence-specific and dosagedependent. Studies suggested that the possible mechanism of antisense oligonucleotides inhibit SRSV mRNA expression involved the attack of the oligonucleotides-RNA hybrid by RNase H activity.Additionally, studies here proved that a single strand DNA produced by asymmetric POR could also inhibit the SRS mRNA expression.