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目的: 构建含人胸腺基质淋巴生成素基因(TSLP)的重组腺病毒载体并表达, 以研究其免疫学功能。方法: 将由人胚肺细胞扩增得到的TSLP基因, 克隆于真核表达载体pcDNA3. 1中, 再亚克隆至穿梭质粒pShuttle中, 并与腺病毒骨架载体pAdEasy -1共同转化大肠杆菌。以获得的重组质粒线性化后转染HEK293细胞, 并包装成病毒颗粒。采用PCR法对重组腺病毒基因进行鉴定, 并以Westernblot检测TSLP蛋白的表达。结果: 通过细菌内同源重组, 成功构建带有人胸腺基质淋巴生成素基因的重组腺病毒质粒, 转染 293细胞后, 包装的重组病毒经PCR检测表明, 基因组含有目的基因,病毒的滴度可达 1×1011pfu/L。Westernblot证实, 感染的肿瘤细胞中有相应基因产物的表达。结论: 通过菌内重组可高效制备带有特定基因的重组病毒。所制备的Ad -TSLP可成功表达相应基因产物, 为进一步研究这一新型细胞因子的功能奠定了基础。
Objective: To construct recombinant adenovirus vector containing human thymic stromal lymphangiogenin (TSLP) gene and study its immunological function. Methods: TSLP gene amplified from human embryonic lung cells was cloned into the eukaryotic expression vector pcDNA3.1, subcloned into the shuttle plasmid pShuttle and transformed into E. coli with the adenovirus backbone vector pAdEasy-1. The resulting recombinant plasmids were linearized and transfected into HEK293 cells and packaged into viral particles. The recombinant adenovirus gene was identified by PCR and the expression of TSLP protein was detected by Western blot. Results: Recombinant adenovirus plasmid with human thymus lymphangiogen gene was constructed successfully by bacterial homologous recombination. After transfected into 293 cells, the recombinant virus was packaged and tested by PCR. The titer of the virus was Up to 1 × 1011pfu / L. Westernblot confirmed the expression of the corresponding gene product in infected tumor cells. Conclusion: Recombinant virus with specific gene can be efficiently prepared by intracellular recombination. The prepared Ad-TSLP can express the corresponding gene product successfully, which lays the foundation for further studying the function of this new cytokine.