重组复制缺陷型腺病毒AdIL-10构建及其在HSC中的表达

Construction of Replication-Deficient Recombinant Adenovirus-Interleukin-10 and its Expression

目的利用AdEasy系统构建重组复制缺陷型腺病毒AdIL 10 ,并观察其在肝星状细胞 (HSC)中的表达。方法从SD大鼠脾脏单核细胞中抽提总RNA ,通过RT PCR获得鼠IL 10cDNA片段 ,插入穿梭质粒pAdTrack巨细胞病毒 (CMV)启动子下游 ,构建重组穿梭载体pAdTrack CMV IL 10 ,线性化后与AdEasy 1电穿孔共转化BJ5 183,获得重组腺病毒质粒pAdIL 10。pAdIL 10用Lipo fectamin包装转染HEK 2 93细胞 ,获得重组腺病毒AdIL 10。将AdIL 10体外感染HSC ,3d后抽提HSC总RNA ,以RT PCR检测IL 10的表达。结果通过酶切和测序法筛选出pAdIL 10 ;经 2 93细胞包装 ,3d后观察到绿色荧光蛋白 (GFP)表达 ;AdIL 10感染HSC3d后观察到GFP表达 ,通过RT PCR可检测到IL 10的表达。结论利用AdEasy系统可制备重组腺病毒AdIL 10 ;AdIL 10感染HSC后可表达IL

Objective To construct recombinant replication-deficient adenoviruses carrying rat IL-10 cDNA by using AdEasy system and to investigate its expression in hepatic stellate cells(HSC). Methods Total RNA was extracted from mononuclear cells of SD rat spleen, and cDNA of IL-10 was obtained using reverse transcriptional polymerase chain reaction(RT-PCR) amplification. The shuttle plasmid-pAdTrack-CMV-IL-10 in which IL-10 cDNA was inserted into the downstream of CMV promoter was established by ligation. Then the linearized shuttle plasmid was cotransferred into colibacillus BJ5183 with adenovirus vector AdEasy-1 to generate the recombinant replication-deficient adenovirus plasmid pAdIL-10 through homologous recombination. The linearized pAdIL-10 was transferred into HEK 293 cell by lipofectamin for packing, and the recombinant replication-deficient adenovirus, AdIL-10, was finally obtained. HSCs infected with AdIL-10 were cultured for 3 d. Total RNA was isolated from HSCs and IL-10 mRNA expressions were detected using RT-PCR. Results The pAdIL-10 was confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed on the third day after packing the linearized pAdIL-10 in 293 cells. GFP expression could be observed on the third day after HSCs infected with AdIL-10, and IL-10 mRNA expression could be detected by RT-PCR. Conclusion The replication-deficient recombinant adenoviruses carring rat IL-10 cDNA, AdIL-10, could be generated by AdEasy system. AdIL-10 could infect HSCs with high efficiency and for a long duration, and IL-10 was expressed successfully.