重组AdPD-L1腺病毒的构建表达及鉴定

目的构建程序性死亡配体-1(PD-L1)重组腺病毒载体,以期用于体内实验研究.方法酶切含有小鼠全长PD-L1 cDNA的pcDNA3.1/PD-L1质粒,亚克隆到穿梭质粒pAdtrack-CMV上,在BJ5183细胞内和AdEasy-1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,氯化铯密度梯度离心纯化病毒.PCR、Western blot鉴定AdPD-L1感染293细胞后PD-L1的表达,同时进行野生型腺病毒的检测和PD-L1在混合淋巴细胞反应中的作用.结果测序、酶切证实PD-L1基因重组腺病毒载体构建成功.RT-PCR、Western blot检测AdPD-L1感染的293细胞,均有PD-L1的表达.无野生型腺病毒的产生,PD-L1能显著抑制小鼠混合淋巴增殖反应.结论成功构建了含小鼠PD-L1基因的重组腺病毒载体,这种膜结合性的PD-L1与其相应受体结合后,对T细胞可起到负性调控的作用,为下一步体内基因治疗实验奠定了基础.

Construction, expression and identification of recombinant adenovirus AdPD-L1

Objective To construct a recombinant adenovirus encoding for Programmed Death Ligand -1 (PD-L1) for future gene therapy in vivo. Methods Full-length mouse PD-L1 cDNA linked with an internal ribosome entry site (IRES)-EGFP cassette was subcloned into pAdtrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with AdEasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombined adenovirus DNA was transfected into 293 cells for packaging and amplification of AdPD-L1 virus, which was purified by CsCl density gradient centrifugation. The expression of PD-L1 was monitored by EGFP fluorescence in infected cells. Results After transfection with adenovirus DNA, infectious virus was only produced to cause cytopathic 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 PD-L1 was verified by RT-PCR and Western blotting in 293 cell after infection with AdPD-L1, but not Ad.EGFP, a similarly constructed control virus. AdPD-L1, but not Ad.EGFP, significantly inhibited mouse mixed leukocyte reaction. Conclusion We have successfully constructed a recombinant adenovirus AdPD-L1 that suppresses T cell response in vitro. The virus will be useful to enforce negative co-stimulation via membrane-anchored PD-L1 to the unwanted T cell response in animal models of disease in vivo.