摘要
目的构建细菌内同源重组型的Cub ilin反义RNA腺病毒表达载体并研究其对肾小管上皮细胞Cub ilin蛋白诱导表达的影响。方法PCR扩增大鼠Cub ilin起始编码区767 bp的DNA片段,与T载体连接,测序鉴定连接方向,通过选择反向和同向的双酶切位点与腺病毒穿梭载体pAdtack-CMV同时酶切和连接,构建反向插入(正向插入作为对照)的腺病毒穿梭载体。将Pm eⅠ线性化的腺病毒穿梭载体pAdtack-ACUB和pAdtack-CUB转染包含腺病毒骨架质粒pAdEasy-1的受体菌B J5183(AdEasyTMXL)进行同源重组。用含有卡那霉素的LB平板筛选同源重组阳性的克隆,PacⅠ酶切和测序鉴定后,转染包装细胞(293细胞),经病毒扩增和纯化后,再以OD法和GFP法计算重组腺病毒的滴度。经免疫印迹法分析Cub ilin反义RNA对Cub ilin蛋白质诱导表达的抑制作用。结果成功构建了Cub ilin的反义RNA腺病毒表达载体(pAdEasy-ACUB)和对照正义RNA腺病毒表达载体(pAdEasy-CUB),滴度为2.0×1010pfu/m l、1.8×1010pfu/m l;Cub ilin反义RNA腺病毒表达载体转染可明显抑制大鼠肾小管上皮细胞的Cub ilin蛋白的诱导表达。结论Cub ilin的反义RNA腺病毒表达载体的成功构建以及对肾小管上皮细胞Cub ilin蛋白诱导表达的明显抑制作用,为在基因水平的研究和治疗蛋白尿引起的肾间质损伤奠定了良好的基础。
Objective To construct antisense RNA expression vector of Cubilin using the adenovirus system by homorecombination in E. coli. and study the effects of Cubilin antisense RNA on Cubilin protein inducible expression in renal tubular epithelial cells （RTEC）. Methods After 767 bp DNA fragment of Cubilin gene was obtained through PCR, this DNA was ligated to the vector-pMD 18-T. The antisense RNA expression vector-pAdtack-ACUB and sense RNA expression vector-pAdtack-CUB was obtained by sequencing, digestion and ligation. The plasmid （pAdtack-ACUB, pAdtack-CUB） digested with Pme I was transformed into BJ 5183 bacteria （AdEasy^TMXL）. Positive clones were selected on LB medium with 25 μg/ml kanamycin. After digested with Pac Ⅰ , plasmids were transfected into packing cells -293 cell line. The titer of antisense recombinant adnovirus-pAdEasy-ACUB and recombinant adnovirus-pAdEasy-CUB was detected by OD method and GFP method after amplification and purification. The effects of Cubilin antisense RNA on Cubilin protein inducible expression was analyzed by Western blotting. Results The titer of pAdEasy-ACUB and pAdEasy-CUB were 2. 0 ×10^10pfu/ml, 1.8 ×10^10pfu/ml. Recombinant adenovirus of Cubilin antisense RNA infection significantly inhibited Cubilin protein inducible expression in RTEC. Conclusion The construction of Cubilin antisense RNA expression vector and the inhibitory effect on Cubilin protein inducible expression in RTEC pave the way for the study and the gene therapy of proteinuria disease.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第11期1157-1160,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目(30370534)~~
