荷载LASP-1基因siRNA的条件增殖型腺病毒的构建

Construction of conditionally replicative adenovirus expressing siRNA targeting LASP-1

目的利用腺病毒包装质粒pBHGE3与携带LASP-1 siRNA的穿梭质粒pZD55-LASP-1、pCA13-LASP-1在HEK293细胞内,包装成病毒ZD55-LASP-1和Ad-LASP-1。方法利用Effectene DNA转染试剂将质粒pBHGE3与质粒pZD55-LASP-1、pCA13-LASP-1分别感染HEK293细胞,纯化并扩增病毒。通过PCR鉴定Ad-LASP-1、ZD55-LASP-1是否包含目的基因,E1区是否缺失及有无野生型腺病毒污染。鉴定正确的病毒进行扩增,CsCl梯度离心纯化,TCID50法测病毒滴度。结果PCR鉴定证明ZD55-LASP-1未混杂野生型腺病毒,且包含目的序列LASP-1 siRNA;Ad-LASP-1 E1区缺失,且包含目的序列LASP-1 siRNA。ZD55-LASP-1滴度为4×10^(10) PFU/ml,Ad-LASP-1滴度为2×10^(10)PFU/ml。Western-blot证实ZD55-LASP-1在肿瘤细胞中表达E1A,Ad-LASP-1不表达E1A。结论靶向表达LASP-1 siRNA序列的病毒ZD55-LASP-1重组成功,为肾癌的下一步基因治疗提供了基础。

Objective To construct virus particles of ZD55-LASP-1 and Ad-LASP-1 in HEK293 cells using adenovirus packing plasmid,pBHGE3 and shuttle vectors,pZD55-LASP-1 or pCA13-LASP-1.Methods The HEK293 cells were infected by plasmid pBHGE3 with plasmid pZD55-LASP-1 or pCA13-LASP-1 using Effectene DNA transfection reagent.PCR tests were used to verify the existence of target genes,deletion of E1 segment as well as contamination of wild adenovirus in Ad-LASP-1 and ZD55-LASP-1.The correct virus was identified for amplification and was purified by CsCl gradient centrifugation.The virus titers were measured by 50%tissue culture infectious dose(TCID50).Results ZD55-LASP-1 was confirmed with no wild-type adenovirus contamination by PCR test and contained the target sequence of LASP-1 siRNA.In AD-LASP-1,the E1 region was missing while the target sequence LASP-1 siRNA existed.The titers of ZD55-LASP-1 and AD-LASP-1 were 4×10^(10) PFU/ml and 2×10^(10) PFU/ml,respectively.Western blot indicated that E1A was expressed by ZD55-LASP-1,but not by AD-LASP-1,in tumor cells.Conclusions The recombination of the virus,ZD55-LASP-1 containing targeting LASP-1 siRNA sequence was successful,and provided a basis for further gene therapy of renal cancer.