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背景:树突状细胞是目前已知的最强大的抗原提呈细胞,已经被应用于免疫治疗的研究中。目的:构建Der p 2真核表达载体,并证明能在小鼠骨髓来源的树突状细胞中表达。设计、时间及地点:单一样本观察,实验于2005-05/12在解放军第三军医大学新桥医院全军呼吸疾病研究所完成。材料:C57BL/6小鼠;plambd-Der p 2购自美国HESKA公司;pCI-neo质粒为解放军第三军医大学新桥医院全军呼吸疾病研究所保存。方法:体外分离,培养小鼠骨髓来源树突状细胞。将原核表达质粒plambdDer p 2携带的Der p 2全长cDNA切下,重组到真核表达质粒pCI-neo中,然后在脂质体介导下,将重组质粒转染到小鼠骨髓来源的树突状细胞,以未转染质粒和转染空白载体pCI-neo的树突状细胞为对照。主要观察指标:①pCI-neoDer p 2重组质粒结构鉴定。②并用反转录-聚合酶链反应、Western Blot检测Der p 2mRNA和蛋白表达。结果:测序证实构建的重组质粒中携带了Der p 2的全长cDNA序列,且与Gene Bank序列完全一致。反转录-聚合酶链反应和Western Blot检测结果提示,重组质粒转染的树突状细胞能表达Der p 2 mRNA和Der p 2蛋白。结论:成功构建重组Der p 2基因真核表达载体,其转染树突状细胞后,能有效地表达于树突状细胞中。
BACKGROUND: Dendritic cells, the most powerful antigen presenting cell known to date, have been used in immunotherapy. Objective: To construct Der p 2 eukaryotic expression vector and to prove that it can be expressed in mouse bone marrow-derived dendritic cells. DESIGN, TIME AND SETTING: A single sample observation was performed at the Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, People's Liberation Army from May 2005 to December 12, 2005. Materials: C57BL / 6 mice; plambd-Der p 2 was purchased from HESKA company in the United States; pCI-neo plasmid was deposited by the Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University. Methods: Mouse bone marrow-derived dendritic cells were isolated and cultured in vitro. The full length cDNA of Der p 2 carried by the prokaryotic expression plasmid plambdDer p 2 was cut out and recombined into the eukaryotic expression plasmid pCI-neo. Then, the recombinant plasmid was transfected into the mouse bone marrow-derived tree Dendritic cells were transfected with untransfected plasmids and dendritic cells transfected with blank vector pCI-neo. MAIN OUTCOME MEASURES: ①PCI-neoDer p 2 recombinant plasmid structure identification. ② The expression of Der p 2 mRNA and protein was detected by reverse transcription - polymerase chain reaction and Western Blot. Results: Sequencing confirmed that the constructed recombinant plasmid contained the full-length cDNA sequence of Der p 2 and was identical to the Gene Bank sequence. Reverse transcription - polymerase chain reaction and Western Blot results suggested that the recombinant plasmid transfected dendritic cells could express Der p 2 mRNA and Der p 2 protein. Conclusion: The eukaryotic expression vector of recombinant Der p 2 gene was successfully constructed. After transfected with dendritic cells, it can be efficiently expressed in dendritic cells.