重组VIP腺病毒载体的构建及其在293细胞的表达

Construction,expression and identification of recombinant adenovirus Ad-VIP in 293 cells

目的构建血管活性肠肽重组腺病毒载体,以期用于体内实验研究。方法利用RT-PCR扩增小鼠大脑VIPmRNA,亚克隆到穿梭质粒pAdTrack-CMV,在pAdeasy内同源重组,筛选阳性克隆,酶切、测序鉴定正确,线性化后脂质体法转染293细胞进行包装、扩增,利用报告基因EGFP对病毒滴度进行监测。PCR、免疫荧光鉴定pAdeasy-VIP感染293细胞后VIP基因的表达,ELISA检测重组病毒转染后293细胞上清中的表达。结果测序、酶切证实VIP基因重组腺病毒载体构建成功。RT-PCR,免疫荧光检测pAdeasy-VIP病毒感染的293细胞,均有VIP的表达。与对照组相比,重组病毒转染后293细胞上清中VIP多肽的表达明显增高。结论成功构建了含小鼠VIP基因的重组腺病毒载体,并成功表达VIP多肽。

Objective To construct a recombinant adenovirus encoding vasoactive intestinal peptide （VIP） for future gene therapy in vivo. Methods Full length mouse VIP cDNA linked with an internal ribosome entry site （IRES）-EGFP cassette was subeloned into pAdTraek-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with pAdeasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuelease digestion and further confirmed by sequencing. The recombinant adenovirus DNA was transfected into 293 cells for packaging and amplification of pAdeasy-VIP virus, which was purified by SARTOBIND Membrane Adsorbers. The expression of VIP was monitored by EGFP fluorescence in infected cells. VIP peptide expression on 293 cells was detected by PCR, immunofluorescent method and ELISA. Results After transfected with adenovirus DNA, infectious virus was only produced to cause eytopathic effect in the permissive cell line 293 but not in the non-permissive cell line HeLa, confirming only replication defective but not wild type virus was generated. The specific expression of mouse pAdeasy-VIP was verified by RT-PCR and immunofluoreseent method in 293 cells after infection with pAdeasy-VIP, but not Ad-EGFP, a similarly constructed control virus, pAdeasy-VIP, but not pAd-EGFP, significantly secretes VIP peptide in supernatant. Conclusion We have successfully constructed a recombinant adenovirus pAdeasy-VIP that expresses VIP peptide in vitro.