含HSV-TK基因及IL-18基因复制缺陷型腺病毒载体的构建和鉴定

摘要

Objective:To construct and identify the recombinant adenovirus vector containing TK gene and IL-18 gene resepectively.Method: Replication-deficient adenovirus vector containing AdCMV-TK gene and AdCMV-IL-18 gene were constructed by using DNA recombination;293 cells were transduced by AdCMV-TK gene and AdCMV-IL-18 gene,then DNA of those transduced cells was extracted and PCR was conducted; AdCMV-TK and AdCMV-IL-18 recombinant adenovirus were efficently amplified in 293 cells and the titer of the virus was determined by OD260 assay.Result:the AdCMV-TK gene and AdCMV-IL-18 gene were identified correctly by PCR,the titer of the virus:AdCMV-TK=1.28×109 PFU/mL;AdCMV-IL-18=1.28×109 PFU/mL.Conclusion:Replication-deficient Recombinant Adenovirus Vector Containing HSV-TK gene and IL-18 gene are constructed successfully, it would provide some basic theory and experimental data for the tumor gene therapy.%目的：分别构建含TK及IL-18基因的重组腺病毒载体，并进行鉴定。方法：通过DNA重组技术，构建含有目的基因的复制缺陷型腺病毒AdCMV-TK及AdCMV-IL-18；将上述重组腺病毒液感染293细胞，提取DNA，进行PCR鉴定；将病毒液感染293细胞，扩增病毒，用OD260法测定病毒滴度。结果：经PCR鉴定，鉴定正确的腺病毒命名为AdCMV-TK及AdCMV-IL-18，测定病毒滴度AdCMV-TK=1.28×109 PFU/mL；AdCMV-IL-18=1.28×109 PFU/mL。结论：成功构建了含HSV-TK基因及IL-18基因重组腺病毒载体，为肿瘤的基因治疗提供了理论依据及实验数据。