CD146短发夹RNA真核表达载体的构建与鉴定

摘要

Objective The overexpression of CD146 ia reportedly associated with perineural invasion in salivary adenoid cystic carcinoma. This study was designed to construct and identify the eukaryotic expression vector expressing short hairpin RNA ( shRNA) targeting human CD146. Methods Two pairs of shRNA were designed according to the human CD146 sequence in the Genebank ( NM_006500) following the rules of Tuschl, and then synthesized and cloned into the linearized pGenesil-1 vector. The recombinant vector was confirmed by enzyme digestion analysis and DNA sequencing. Results The inserted sequence of the recombinant pGe-CD146-shRNA vector was exactly demonstrated by double restrictive endonuclease digestion and sequence analysis. Conclusion The pGe-CD146-shRNA eukaryotic expression vectors were successfully constructed and identified.%目的 CD146基因表达增高与涎腺腺样囊性癌(adenoid cystic carcinoma,ACC)嗜神经侵袭(perineural invasion,PNI)的临床症状相关.为进一步研究CD146基因的功能,构建人CD146短发夹RNA(short hairpin RNA,shRNA)真核表达载体并鉴定.方法 根据Genebank中靶基因CD146序列,按照Tuschl设计原则,设计、合成编码其shRNA的互补寡核苷酸序列,克隆至线性化的pGenesil-1载体中并对重组质粒进行酶切分析及测序鉴定.结果 经过限制性内切酶酶切分析和DNA测序鉴定,证实重组质粒pGe-CD146-shRNA的序列正确.结论 成功构建了靶向人CD146基因的shRNA真核表达载体系统,为进一步研究CD146基因的功能奠定了实验基础.