摘要
目的建立９９ｍＴｃ标记反义寡核苷酸（ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ，简称ｏｌｉｇｏ）的方法。方法将联肼尼克酰胺衍生物偶联到人工合成的１５ｍｅｒｃｍｙｃｍＲＮＡ反义ｏｌｉｇｏ末端连接的氨基上，以三羟甲基甘氨酸做协同配体进行９９ｍＴｃ标记，用ＳｅｐＰａｋ反相柱纯化９９ｍＴｃｏｌｉｇｏ，并评价其特性。结果标记率为６０％～８０％，纯化后９９ｍＴｃｏｌｉｇｏ的放化纯度在室温４小时内保持＞９５％，比活度在５～４２ＭＢｑ／μｇ范围。对照组标记到ｏｌｉｇｏ上的放射性＜５％。９９ｍＴｃｏｌｉｇｏ在新鲜人血清中稳定，不被核酸酶降解，也不与血清中蛋白质结合，打点杂交证实９９ｍＴｃ标记的反义探针探测其靶序列（正义链）ＤＮＡ的最大可探测灵敏度为３０ｎｍｏｌ／Ｌ。
Purpose To develop a method of radiolabeling of oligonucleotide (oligo) with ^(99m)Tc for in vivo imaging application Methods 15 mer oligos complementary to cmyc oncogene mRNA transcription start site (antisense) and its complementary strand (sense) were synthesized The antisense strand with a 5′end aminolinker was conjugated with HYNIC as bifunctional chelating agent and then labeled with ^(99m)Tc using Tricine as coligand ^(99m)Tcoligo was purified by SepPak reverse phase column and its properties were evaluated Results The labeling efficiency of ^(99m)Tcoligo ranged from 60% to 80%, the radiochemical purity post SepPak purification remained higher than 95% 4h post labeling at room temperature The specific activity which was dependent on the specific activity of pertechnetate, was 5～42MBq/μg Only less than 5% radioactivity was observed on the oligos in the control labeling ^(99m)Tcoligo is stable when incubated with fresh human serum, no degeneration and no binding with protein were observed The maximum detectable sensitivity of ^(99m)Tcoligo was 30nmol/L when using it to detect target sequence (sense strand) on dot blotting Conclusions ^(99m)Tcoligo labeled with the method developed in this laboratory holds desirable stability and hybridization properties
出处
《中华核医学杂志》
CAS
CSCD
北大核心
1998年第3期175-177,共3页
Chinese Journal of Nuclear Medicine
基金
英国帝国癌症研究基金
关键词
寡核苷酸探针
反义
肿瘤
MRNA
诊断
Oligonucleotide probes Oligonucleotides, antisense RNA, messengerTechnetium
