Der p 2基因真核表达载体构建及其在小鼠树突状细胞中的表达(英文)

Construction of eukaryotic expression vector of Der p 2 gene and its expression in mouse dendritic cells

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 potent antigen presenting ceils known at present, have been extensively used in the immunotherapy as adjuvant. OBJECTIVE： The present study was to construct Der p 2 eukaryotic expression vector and validate its expression in the mouse bone marrow-derived dendritic cells. DESIGN, TIME AND SETTING： A single sample observation was performed at the Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University of Chinese PLA between May and December 2005. MATERIALS： C57BL/6 mice were included. Plambd-Der p 2 was the product of Heska Company, USA. pCI-neo plasmid was provided by the Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University of Chinese PLA. METHODS： Mouse bone marrow-derived dendritic cells were in vitro isolated and cultured. Complete Der p 2 cDNA was spliced from prokaryotic expression vector plambd-Der p 2, and then cloned into eukaryotic expression vector pCI-neo （pCI-neo-Der p 2）. The positive recombinants pCI-neo-Der p 2 transfected into dendritic cells. Non-transfected and blank vector pCI-neo-transfected dendritic cells were used as controls. MAIN OUTCOME MEASURES： （1）Identification of pCI-neo-Dcr p 2 recombinant plasmid. （2）Detection of Der p 2 mRNA and protein expression by reverse transcription-polymerase chain reaction （RT-PCR） and Western Blot techniques. RESULTS： Sequencing results showed Der p 2 cDNA in pCI-neo-Der p 2 was in coincidence with the sequence registrated in Gene Bank. RT-PCR and Western Blot results showed that expression of Der p 2 mRNA and protein could be detectable in the pCI-neo-Der p 2-transfected dendritic ceils. CONCLUSION： The Der p 2 cDNA was successfully constructed into the eukaryotic expression vector, and Der p 2 gene and protein could be expressed efficiently in dendritic cells.