HJURP基因缺陷表达的人胚胎绒毛细胞模型的构建与鉴定  

Construction and identifi cation of a model for HJURP gene defect expression in human embryo villus cells

作　　者：薛慧琴[1] 高庭[1] 朱镭[1] 孙夏瑜[1] 卢洪涌[1] 张玉萍[1] 周岩[1] 燕美琴[1] 祁春茹[1] 

机构地区：[1]山西省儿童医院遗传室山西省妇幼保健院,太原030013

出　　处：《中国优生与遗传杂志》2015年第11期27-31,共5页Chinese Journal of Birth Health & Heredity

基　　金：山西省卫计委青年基金项目(编号:201201012)

摘　　要：目的构建HJURP基因的RNA干扰慢病毒表达载体并在人胚胎绒毛细胞上鉴定其沉默效率,进而提供HJURP基因缺陷表达的原代胚胎绒毛细胞模型。方法针对目的基因HJURP的序列,按照RNA干扰序列的设计原则,设计合成3个HJURP基因特异性的小分子干扰RNAi靶点,将其合成短发夹RNA(sh RNA)并退火形成双链RNA,克隆至慢病毒载体中形成sh RNA表达载体,通过PCR和测序鉴定获取正确重组载体。将筛选出的重组载体与慢病毒包装质粒共转染293T细胞并测定病毒滴度。随后将其干扰人胚胎绒毛细胞,采用Western blotting和real-Time PCR检测靶基因的沉默效率。结果对重组载体p HBLVU6-Zs Green-Puro进行测序鉴定,证实插入的sh RNA序列无碱基变异。重组慢病毒载体经293T细胞包装后,病毒滴度为108PFU/ml。RNA干扰人胚胎绒毛细胞HJURP基因后,可见感染后内源HJURP的蛋白表达水平显著下降,HJURP m RNA水平明显下降,其中sh RNA1干扰效率大于70%。结论成功构建了HJURP基因的sh RNA慢病毒表达载体,该重组载体可以在细胞水平有效沉默靶基因,为后续研究HJURP基因表达抑制对人胚胎绒毛细胞的增殖凋亡和染色体分离的影响,探索HJURP基因在自然流产发生中的作用机制提供细胞模型。Objective：To construct a lentiviral vector for RNA interference（RNAi）of the HJURP gene and to identify the silencing efficiency in the human embryo villus cells.To provide a human embryo villus cells multiplication and chromosome segregation. Methods：In accordance with the study,3 specific sequences of si RNA targeting HJURP gene were designed,synthesized,then the complementary DNA containing both sense and antisense oligonucleotides of the targeting sequences were annealed and inserted into the lentiviral vector.The correct clonings were confirmed by PCR and sequencing.The most effective recombinant lentivirus vector was screened,and the recombinant plasmids with the lentivirus packaging mixes were co-transfected into 293 T cells to obtain packaged lentivirus particles.Then viral titer was determined.The silencing efficiency of target gene in human embryo villus cells was detected by Real-Time PCR.Results：DNA sequencing showed that the sh RNA sequence was successfully inserted into the lentivirus vector.The recombinant lentiviral vector was successfully transfected into 293 T cells.The recombinant lentivirus had a titer of 108 PFU/ml.After silencing HJURP gene in human embryo villus cells,the expression level of HJURP m RNA decreased significantly,and the RNAi efficiency was greater than 70%. Conclusion：A lentiviral sh RNA expression vector targeting the HJURP gene is successfully constructed and may effectively silence the target gene at a cellular level,which provides a experimental model for the influence of HJURP gene expressing inhibition on human embryo villus cells multiplication and chromosome segregation.

关 键 词：人类胚胎绒毛细胞 HJURP RNA干扰 慢病毒 

分 类 号：R714.21[医药卫生—妇产科学] R394[医药卫生—临床医学]