人血小板源性生长因子A基因发夹结构小干扰RNA真核表达载体的构建及鉴定

Establishment and identification of small hairpin interfering RNA eukaryotic expression vectors of human platelet-derived growth factor A

目的:血小板源性生长因子对诱发视网膜色素上皮细胞移行、最终导致增生性玻璃体视网膜病变起到了重要作用。构建人血小板源性生长因子A具有发夹结构小干扰RNA(shRNA)表达质粒,以观察其对人血小板源性生长因子A基因表达的沉默效果。方法:实验于2007-04/2007-07在吉林大学第一临床医院传染科研究室完成。根据血小板源性生长因子A基因mRNA序列,设计有小发夹结构的3条寡核苷酸序列,克隆到空载体pGPU6/GFP/Neo中,构建重组质粒,同时设计构建分别针对人GAPDH干扰质粒作为阳性对照,不针对任何特异基因的质粒作为阴性对照。通过酶切鉴定和测序验证阳性克隆。结果:经重组质粒筛选,重组质粒测序鉴定与设计的shRNA转录模板序列相同,均证实重组质粒构建成功。结论:利用RNA干扰技术路线可成功构建靶向人血小板源性生长因子A的发夹结构小干扰RNA表达载体。

AIM： Platelet-derived growth factor （PDGF）-A plays an important role in leading to proliferative vitreous retinopathy through inducing the migration of retinal pigment epithelium. This study was designed to establish the recombinant plasmid of small hairpin interfering RNA （shRNA） against human PDGF-A and observe silence effect of the human PDGF-A expression. METHODS： The experiment was conducted at the Infection Laboratory of the First Affiliated Hospital of Jilin University from April to July in 2007. Recombinant plasmid was constructed based on human PDGF-A mRNA sequence. Three pairs of oligo nucleotides were synthesized and inserted into plasmid pGPU6/GFP/Neo to generate shRNA eukaryotic expression vectors. Interfering plasmid from GAPDH were established as positive control and interfering plasmid targeting none gene served as negative control. The recombinant were verified by enzyme digestion and sequencing. RESULTS： The recombinant plasmid sequence was the same as the sequence of shRNA transcribed sequence. So the recombinant vectors were established successfully. CONCLUSION： The shRNA expression vector targeting human PDGF-A can be established successfully by RNA interference technique.