人白介素24腺病毒载体构建及其生物学活性分析

Construction recombinant adenovirus vector expressing human interleukin-24 and analysis of its biological activity in vitro

目的:克隆人白介素24基因,构建其腺病毒载体,获取病毒重组子,并研究其生物学活性。方法:用密执毒素(Mezerein)诱导HeLa细胞表达IL-24,通过RT-PCR获取IL-24 cDNA,将其亚克隆至pAdTrack-CMV载体,经PmeⅠ线性化后,与腺病毒的骨架载体pAdEasy-1在BJ5183菌中同源重组,HEK293细胞包装扩增,PCR、Western blot鉴定。AdIL-24处理宫颈癌CaSki细胞,通过MTT细胞存活实验检测其活性;Hoechst33342染色、流式细胞仪检测凋亡;Western blot检测Bax、Bcl-2、p53蛋白的表达。结果:获取人IL-24的cDNA,序列与GeneBank公布序列完全一致,成功构建腺病毒载体,AdIL-24对CaSki细胞具有抑制生长、促进凋亡的作用,并上调Bax、p53蛋白表达,下调Bcl-2蛋白表达。结论:成功构建人IL-24的重组腺病毒载体,其病毒重组子具有生物活性。

Objective： To clone human IL-24 gene, construct its recombinant adenovirus vector, and to study its biological activity.Methods：The expression of IL-24 in HeLa cells was induced by mezerein,IL-24 cDNA was amplified by RT-PCR and then was subcloned to the vector of pAdTrack-CMV. Generation of recombinants by cotransforming the Pine Ⅰ -cut shuttle plasmids with pAdEasy-1 backbone vectors in BJ5183,virus was multiplied in 293 cells and was identified by PCR and Western blot.The inhibitory effect of IL-24 to CaSki cells was detected by MTr assay and cell survival analysis. Apoptosis was detected by Hoechst 33342 stain and flow cytometry. The expression of Bax,Bcl-2 and p53 was detected by Western blot.Results：The sequence of IL-24 gene was identical with that in GeneBank, the recombinant adenovirus vector was successfully constructed, this study showed that AdIL-24 induced growth suppression and apoptosis of CaSki cells. The Bax and p53 protein were up regulated while Bcl-2 protein was down regulated. Conclusion： The adenovirus vector of hIL-24 was successfully constructed and adenovims recombinants had biological activity.