摘要
目的构建晶状体来源的上皮生长因子p52(lens epithelium derived growth factor p52,LEDGFp52)基因RNA干扰(RNA interference,RNAi)的真核细胞表达载体。方法以LEDGFp52为靶基因,以pGensil-1质粒为载体,设计构建重组体,根据GenBank数据库提供的LEDGFp52基因核苷酸序列,按照Tuschl设计原则,选择设计2条带发夹结构的核苷酸序列,克隆到空载体pGensil-1中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析。结果经酶切鉴定筛选出的重组体测序结果与目的序列完全一致,重组载体构建成功,重组质粒转染HeLa细胞48h,Western blotting检测到LEDGFp52蛋白表达的改变。结论利用RNAi技术可成功构建抑制LEDGFp52表达的小干扰RNA重组体。
Objective To construct the eukaryotic expression vector of lens epithelium derived growth factor p52 （ LEDGFp52 ） of RNA interference（RNA1）. Methods Recombinant were designed and established by target gene LEDG- Fp52 mid plasmid pGensfl-1 based on LEDGFp52 eDNA sequences of genomes, two pairs of oligonucleotides were synthesized based on the design principle of Tusehl and inserted into plasmid pGensfl-1 to generate siRNA eukaryotic expression vector,DH5α strains were transformed, plasmids were extracted, and recombinant vector were identified and analyzed by the restrictive endonuclease digestion map and sequence analysis. The recombinant plasmid （pGensil-1- LEDGFp52） was transfected into the cultured HeLa cells. At 48 hours after transfection,the whole cell protein was extracted,and the protein level was.detected by Western blotting with mouse-anti-human LEDGFp52 monoclonal antibody. Results Recombinant plasmids were completely coincided with the designs by the restrictive endonuclesse digestion map and the sequence analysis, the protein level of LEDGFp52 was down-regulated at 48 hours after transfected pGensil-1-LEDGFp52 expression vector into HeLa cells, the recombinant eukaryotic expression vector were constructed successfully. Conclusion siRNA recombinant can be constructed successfully by RNAi technique to inhibit the expression of LEDGFp52. [ Rec Adv Ophthaimol 2007 ;27 （3） ：184-187 ]
出处
《眼科新进展》
CAS
2007年第3期184-187,共4页
Recent Advances in Ophthalmology
关键词
晶状体来源的上皮生长因子p52
RNA干扰
真核表达载体
构建
鉴定
lens epithelium derived growth factor p52
RNA interference
eukaryotic expression vector
construction
identification
