萤火虫荧光素酶基困的反义RNA及其缺失片段阻抑基因表达的研究

利用离体转录载体ｐＳＰ１８、ｐＧＥＭ－３Ｚ和萤火虫荧光素酶基因（Ｌｕｃ）及其缺失片段构建了一组融合质粒。以这些质粒为模板，通过离体转录产生荧光素酶基因的ｍＲＮＡ，反义ＲＮＡ及大小和结构不同的反义ＲＮＡ缺失片段，再通过显微注射分别将其引入爪赡卵母细胞的细胞质。注射后的卵母细胞经一定时间培育，然后检测其翻译产物一荧光素酶的相对活性。结果表明，荧光素酶基因的表达受反义ＲＮＡ及其缺失片段的严重阻抑，表达抑制和反义ＲＮＡ之间存在明显的剂量效应。而且，基因表达的抑制程度主要的和反义ＲＮＡ片段的结构有关。

人绒毛滋养细胞单克隆抗体制备及初步鉴定

采集６～８周人工流产胎盘绒毛，经胶原酶处理及梯度离心后得到滋养细胞，再用ＮＰ－４０提取滋养细胞抗原。经常规免疫、细胞融合和克隆化后得到２０多株单克隆抗体（ＭｃＡｂ）细胞株。其中１０株ＭｃＡｂ进行了免疫学鉴定。免疫球蛋白分类，２株为ＩｇＭ，８株为ＩｇＧ，其中４株为ＩｇＧｌ。免疫斑点试验结果显示，１０株ＭｃＡｂ均为阳性，其滴度为１：８０～１：１２８０。免疫荧光定位，１０株中７株与滋养层细胞反应，２株与绒毛基底膜结合，１株为阴性。并对其进行了免疫转移印迹试验和交叉反应性研究，初步研究结果显示，单抗Ｔ６有着较好的特异性，值得进一步研究。

An Accurate Model for the Expression of the Antisense RNA Block Gene:the Firefly Luciferase Gene-Xenopus Oocyte System

The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.

反义RNA阻抑基因表达的精确模型——萤火虫荧光素酶基因-爪蟾卵母细胞系统

80年代建立的“反义基因操作技术”不仅打破了通过基因突变认识基因的局限,成为了解基因功能新的有效工具.而且,还拓宽了借助基因操作治疗疾病和改良植物品质的应用途径。虽然,至今仍未取得真核细胞能利用自身存在的反义RNA调节基因表达的证据,但是,人工构建的反义RNA或反义基因能有效地抑制细胞内源或外源基因的暂时性或稳定性表达已有越来越多的报道.只是对抑制机理,最适抑制条件等仍了解很少.