CEA启动子驱动下表达Hsp70基因的重组腺病毒的构建及鉴定

目的 构建癌胚抗原(CEA)启动子驱动下靶向表达热休克蛋白70 (Hsp70)基因的重组腺病毒。方法 通过RT-PCR和PCR的方法分别扩增人Hsp70基因和CEA启动子，构建穿梭质粒pDC316-pCEA-Hsp70。将所得的穿梭质粒与腺病毒骨架质粒共转染293细胞获得重组腺病毒Ad5-pCEA-Hsp70。通过梯度离心纯化病毒，采用半数细胞培养物感染量测定病毒滴度。分别以重组的腺病毒感染CEA阳性的人胰腺癌BxPC3细胞和CEA阴性的SW1990细胞，通过RT-PCR和ELISA法检测Hsp70 mRNA和蛋白的表达。结果 酶切和测序证实Hsp70基因和CEA启动子成功插入PDC316质粒。与骨架质粒共转染293细胞获得的病毒经PCR扩增证实重组腺病毒构建成功。最终获得的病毒颗粒数达2.2 ×1011 vp/ml，滴度为1.5×1010 PFU/ml。重组腺病毒感染CEA阳性表达的BxPC3细胞后，可显著增加Hsp70 mRNA和蛋白的表达，而感染CEA阴性的SW1990细胞，Hsp70 mRNA和蛋白的表达无变化。结论 成功构建了能够在CEA阳性细胞中靶向表达Hsp70基因的重组腺病毒Ad5-pCEA-Hsp70，为进一步研究奠定了基础。

Construction and identification of recombinant adenovirus containing heat shock protein70 gene driven by carcinoembryonic antigen promoter

Objective To construct the recombinant adenoviros containing heat shock protein70 (Hsp70) gene driven by carcinoembryonic antigen (CEA) promoter. Methods Hsp70 gene and CEA promoter were amplified by RT-PCR and PCR, and then subcloned into the shuttle vector pDC316 to construct the recombinant vector PDC316-pCEA-Hsp70. The recombinant vector was co-transfected with adenoviral backbone plasmid into HEK293 cells to generate the recombinant adenovirus Ad5-pCEA-Hsp70. The recombinant adenovirus was purified by CsCl banding and titrated by 50％ tissue culture infective dose (TCID50) assay. After transfection of the recombinant adenovirus into human pancreatic cell lines SW1990 and BxPC3, the expression of mRNA and protein level of Hsp70 were determined by RT-PCR and ELISA,respectively. Results Digestion and DNA sequencing certified that the Hsp70 gene and CEA promoter was successfully inserted into pDC316 plasmid. Virus acquired through co-transfection with backbone plasmid was confirmed to be constructed successfully by PCR amplification. The particles finally expressed was 2.2 ×1011vp/ml, and the titer was 1.5 x 1010 PFU/ml. BxPC3 cancer cells with positive CEA expression showed increased expression of Hsp70 mRNA and protein after infected by recombinant adenovirus; while SW1990 cancer cells with negative CEA expression showed no change of expression of Hsp70 mRNA and protein after infected by recombinant adenovirus. Conclusions The recombinant adenovirus Ad5-pCEA-Hsp70 which can express Hsp70 gene in CEA positive cancer cells is constructed successfully.