CD105特异性shRNA表达质粒的构建和筛选

Construction and screening of shRNA expression plasmids targeting CD105

目的：使用Pgenesil-1质粒构建CD105RNA干扰重组体，在激光诱导BN大鼠脉络膜新生血管模型中，筛选高效抑制CD105基因表达的shRNA。方法：根据CD105基因序列信息设计了3条shRNA以及1条非特异阴性序列，利用含U6启动子的表达质粒Pgene-sil-1构建其RNA干扰重组体。采用视网膜下注射的方法使shRNA表达质粒转染大鼠视网膜和脉络膜，观察质粒所携带绿色荧光蛋白基因表达情况。根据不同的表达质粒对BN大鼠CNV模型2wk时CD105基因mRNA表达的抑制效果与空白对照及仅加转染试剂组比较分析，筛选有效的抑制序列。结果：转染后1d，在荧光显微镜下可见有明显的绿色荧光分布于实验眼视网膜全层，包括RPE层。2～3wk荧光强度比1wk增强，并持续表达4wk。空白对照眼无绿色荧光表达。构建了3条CD105shRNA和阳性对照Pgenesil—HK，基因测序证实目的序列成功插入质粒Pgenesil-1中。研究发现不同的质粒转染后对CD105在CNV高峰期的表达干预效果不同。Pgenesil-eng2和Pgenesil-eng3可明显抑制CNV模型在2wk时CD105mRNA的表达，抑制效率分别为（78±5）％（P〈0．01）；（52±3）％（P〈0．01）。转染Pgenesil-engl，Pgenesil-HK组及仅加转染试剂组与空白对照组相比CD105mRNA表达水平无明显变化。结论：在阳离子脂质体辅助下，Pgenesil-1质粒可以成功地将目的基因转染至大鼠视网膜各层次，且表达强度高，时间长于4wk。Pgenesil-eng2能明显抑制BN大鼠视网膜组织CD105mRNA的表达。

AIM： To construct the recombinant plasmids expressing CD105 short hairpin RNA （shRNA） by Pgenesil-1 plasmid, and to screen the highly efficient shRNA in Brown Norway （BN） rat CNV model. METHODS： Metafectene containing Pgenesil-1 was injected into subretinal space in the eyes of BN rat. Localization of GFP was observed by fluorescence microscopy. Four recombinant Pgenesil-1 with U6 promoter were constructed, 3 targeting rat CD105 and 1 unspecific. Finally, the plasmids were identified by sequence analysis. Three recombinant plasmids being transfect into BN rat CNV model, the expression of CD105 mRNA level was determined by reverse transcription polymerize chain reaction 2 weeks later. RESULTS： One day after transfection, green fluorescence was observed in the rat＇s retina including RPE ceils under fluorescent microscope. The intensity became stronger on the second and third week than that on the first week, and sustained for 4 weeks. The CD105 shRNAs were successfully inserted into plasmid Pgenesil-1. The recombinants were identified by sequencing. Compared with the controls, the expression of CD105 mRNA level in BN rat CNV model transfect with CD105 shRNA recombinants was markedly down-regulated （78 ± 5）% （ P〈 0.01 ） and （52 ±3）% （ P 〈 0.01 ） respectively in two groups of recombinant plasmids, whereas it had not any significant changes in another shRNA and unspecific shRNA transfect and only with transfection reagent to BN rat CNV model. CONCLUSION： Metafectene can transfect Pgenesil-1 into the retina of the BN rat effectively, and their expression can maintain 4 weeks after transfection. Three recombinants expressing CD105 shRNA are successfully constructed, shRNA sequences potently inhibiting CD105 are screened out successfully too, which lends itself to new gene therapy for CNV.