乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒的构建及鉴定

目的：构建乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。方法：乙酰肝素酶cDNA全长扩增和最佳siRNA干扰片段筛选分别采用PCR和Real-time PCR方法,慢病毒系统载体分别使用pWPI和siRNA pSico系统,采用限制性内切酶快速连接方法联接目的基因和最佳最佳siRNA干扰片段,表达载体鉴定均采用核苷酸序列测定,HPSE重组慢病毒表达质粒和siRNA片段细胞转染采用脂质体转染法。结果：成功扩增乙酰肝素酶全长并连接入pWPI载体构建成重组表达载体HPSE-pWPI,重组质粒测序结果显与HPSE基因的同源性达99%。转染293T后有HPSE基因的表达。筛选出最佳siRNA干扰片段为HPSE-1222并成功插入pSico载体,构建成重组表达载体HPSE-siRNA pSico,重组载体测序显示与构建的shRNA结构序列完全一致。结论：成功采用慢病毒载体系统构建了乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。

Construction For Recombinant HPSE Lentivirus Transgenic And Interference Plasmid

Objective： To construct recombinant HPSE transgenic lentivirus and interference plasmid.To investigate the molecular mechanism of HPSE in tumor invasion and metastasis. Methods： The amplifion of full-length heparanase eDNA fragment and the best siRNA interference filters were carried out by PCR and Real-time PCR method.The lentiviral vector plasmid was used by siRNA pSico and pWPI system srespectively. The restriction enzyme quiekconnection method was used to join the target genes and the best best siRNA interference fragment with plasmid. The identification of expression vector was used by nucleotide sequence analysis. The cells were transfected with HPSE recombinant plasmid and siRNA fragments by using liposome transfection method. Results： The full-length of heparanase was successfully amplified and connected into the pWPI vector. The recombinant HPSE gene sequencing results significant homology with the 99%. There was postitive expression of HPSE gene in 293T cells transfected by recombinant HPSE gene plasmid.The best siRNA interference fragment selected was HPSE-1222, and successfully inserted into pSico vector. Recombinant vector sequence displays exactly the same sequence of shRNA structure. Construction： The heparanase recombinant lentiviral transfer and siRNA interference plasmids were constructioned Successfully.lt would be to lay the foundation for investigatig the molecular mechanism of HPSE in tumor invasion and metastasis.