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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 RNA interference lentivirus expression vector of HJURP gene and identify its silencing efficiency on human embryonic villi cells to provide a primary embryonic villus cell model with defective expression of HJURP gene. Methods According to the sequence of target gene HJURP, three small interfering RNAi targets of HJURP gene were designed and synthesized in accordance with the design principles of RNA interference sequence. The target RNA was synthesized and annealed to form double stranded RNA. Cloned into lentiviral vector to form sh RNA expression vector and confirmed by PCR and sequencing to obtain the correct recombinant vector. 293T cells were co-transfected with the recombinant vector and the lentivirus packaging plasmid and the virus titer was determined. Subsequently, they were interfered with human embryonic villus cells, and the silencing efficiency of the target gene was detected by Western blotting and real-time PCR. Results The recombinant vector p HBLVU6-Zs Green-Puro was sequenced and confirmed that there was no base variation in inserted sh RNA sequence. Recombinant lentiviral vector 293T cell packaging, the virus titer of 108PFU / ml. After RNA interference of HJURP gene in human embryonic chorionic villus cells, the expression of endogenous HJURP protein decreased significantly and the level of HJURP m RNA decreased obviously. The interference efficiency of sh RNA1 was more than 70%. Conclusion The recombinant lentiviral vector of shuRNA gene of HJURP gene was successfully constructed. The recombinant vector can effectively silence the target gene at the cellular level. In order to further study the effect of HJURP gene expression on the proliferation, apoptosis and chromosome segregation of human embryo villus cells, The role of genes in the pathogenesis of spontaneous abortion provides a cellular model.