I型内含子核酶介导靶向CEA双报告基因显像的体外研究

In vitro study of type I intron-mediated dual reporter gene imaging for carcinoembryonic antigen

目的构建靶向CEA的以I型内含子核酶为载体核心介导的生物发光一核素双报告基因显像系统。方法采用基因工程技术构建靶向CEA的I型内含子核酶及核酶-荧光素酶-胸苷激酶（Rib53-Fluc-tk）报告质粒，采用细胞生物发光等方法检测核酶反式剪接产物的表达；以Iodogen固相氧化法合成131I-氟-碘阿糖呋喃基尿嘧啶（FIAU），行细胞摄取实验。采用两样本t检验、方差分析及最小显著差异（LSD）t检验进行统计分析。结果Rib53一Fluc—tk测序正确；乳腺癌细胞MCF-7的荧光素酶比值增高，可达O．64±0．10（n=4）；MCF-7细胞转染Rib53-Fluc—tk后的反式剪接产物条带大小为520bp，测序结果正确；pCDNA3．1-CEA及Rib53-Fluc-tk共转人胚。肾细胞293T，可探测到生物发光信号。131I—FIAU的标记率为（64．02±4．79）％（n=3），放化纯为（95．96±1．07）％（n=3），标记产物在人血清中24h的稳定性高。293T单独转染Rib53-Fluc-tk，细胞摄取率仅为（0．31±0．01）％（n=4）；293T共转染pCDNA3．1-CEA、Rib53-Fluc-tk质粒，细胞摄取率增加至（1．40±0．06）％（n=4），F=1007．29，t=136．34，P〈0．01。结论成功构建了I型内含子核酶为核心介导的靶向CEA的双报告基因系统，并对CEAmRNA进行了生物发光以及细胞摄取的检测，为改善传统报告基因显像的特异性奠定了体外实验的基础。

Objective To develop a specific trans-splicing intron ribozyme type I -mediated dual reporter gene system （Rib53-Fluc-tk） for targeting CEA. Methods The novel CEA-targeting trans-splicing ribozyme with the downstream reporter system （Rib53-Fluc-tk） was constructed by genetic engineering technology. The trans-splicing reaction product was evaluated using the lzlI-5-iodo-2＇-fluro-l-beta-D-arabino- furanosy-luracil （FIAU） cellular uptake rates and the bioluminescence. Two-sample t test, the analysis of variance and the least significant difference （LSD） t test was performed for data analysis. Results The se- quence of Rib53-Fl^-tk was proved by gene-sequencing test. Human MCF-7 breast cancer cells showed a high ratio of firefly luciferase/renilla luciferase （ 0.64±0.10, n = 4）. A 520 bp band of product existed, which matched with the predicted size using RNA from cells transfected with Rib53-Fluc-tk in MCF-7. Sig nals were detected by bioluminescence in human embryonic kidney 293T cells cotransfected with Rib53- Fluc-tk and pCDNA3.1-CEA. The labelling rate of 131I-FIAU was （64.02±4.79）% （n=3）. The radiochemical purity was （95.96± 1.07）% （n = 3）, and the stability of the radiocompound remained high in human serum at least for 24 h. The uptake of 131I-FIAU in 293T ceils transfected with Rib53-Fluc-tk was （0.31±0.01）% （n= 4）, while it increased with the incubation time in 293T ceils eo-transfected with pCDNA3. I-CEA and Rib53-Fluc-tk and reached （1.40±0.06） % at 4.5 h （ F= 1 007.29, t = 136.34, both P〈0.01）. Conclusions A novel and specific reporter gene in the cellular level was established. Taking advantage of trans-splieing reaction of the ribozyme, it could improve the specificity of the reporter gene imaging.