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目的:构建携带DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)RNAi基因的重组杆状病毒载体,以进一步研究DNMT1对胰腺癌相关基因的影响。方法:首先将带有BglⅡ和HindⅢ酶切黏性末端的60 bp正义和反义寡核苷酸退火,与用BglⅡ和HindⅢ双酶切后的线性化载体pSUPER连接构建成pSUPER si-DNMT1-D,然后将H1启动子和RNAi序列从上述重组载体切下,插入至同样双酶切的pFastBac1载体,经过转座重组,抽提重组BacmidDNA,转染草地贪夜蛾细胞Sf9,获得重组杆状病毒Bac-si-DNMT1-D。将上述重组杆状病毒感染胰腺癌PaTu8988细胞系,其中以重组杆状病毒r-Bac-CMV-EGFP作为阴性对照。然后运用荧光定量PCR检测DNMT1基因表达变化。结果:重组杆状病毒能够感染PaTu8988细胞,并且能显著干扰DNMT1基因表达。结论:杆状病毒表达系统可以作为RNAi载体,为以后的基因治疗开辟新的途径。
Objective: To construct a recombinant baculovirus vector carrying DNA methyltransferase 1 (DNMT1) RNAi gene to further investigate the effect of DNMT1 on pancreatic cancer related genes. Methods: The 60 bp sense and antisense oligos with BglII and HindIII digestion cohesive ends were annealed with the linearized vector pSUPER digested with BglII and HindIII to construct pSUPER si-DNMT1-D , And then H1 promoter and RNAi sequences were cut from the recombinant vector and inserted into the same double-digested pFastBac1 vector. After transposon recombination, recombinant BacmidDNA was extracted and transfected into Sf9 of S. frugiperda to obtain recombinant baculovirus Bac-si-DNMT1-D. The recombinant baculovirus was used to infect pancreatic cancer PaTu8988 cell line, and recombinant baculovirus r-Bac-CMV-EGFP was used as a negative control. Fluorescent quantitative PCR was then used to detect the DNMT1 gene expression. Results: The recombinant baculovirus could infect PaTu8988 cells and could significantly interfere with DNMT1 gene expression. Conclusion: Baculovirus expression system can be used as RNAi vector to open up new avenues for future gene therapy.