摘要
目的构建重组真核表达质粒PTT3-FLT3L—hIgGFc，鉴定其诱导机体表达蛋白FLT3L—hIgGFc的水平以及诱导小鼠淋巴结脾脏树突状细胞（DCs）的功能，探索体内注射质粒代替注射蛋白诱导DCs方法的可行性。方法用PCR方法从小鼠肥大细胞1~815细胞系中扩增出FLT3L基因片段，连接到P＇133-hIgGFc载体上，得到重组真核表达质粒PTF3-FLT3L，hIgGFc；通过酶切鉴定和测序检测目的基因核苷酸序列；尾静脉高压注射PTT3．FLT3L—hIgGFc质粒和PTT3-hIgGFc对照质粒，9d后取小鼠淋巴结和脾脏，流式细胞术检测CD3、CD19、DX5、CD11c、MHC-Ⅱ等的表达来鉴定DCs的比例和数量。结果酶切和测序结果显示PTT3-FLT3L—hIgGFc质粒构建成功。小鼠尾静脉高压注射PTr3-FLT3L—hIgGFc后，血清中持续含有高于生理水平的FL33L—hIgGFc蛋白，小鼠脾脏体积明显增大，淋巴结和脾脏中CD11^＋MHC-Ⅱ^＋DC明显增多。结论成功构建了PTT3-FLT3L—hIgGFc重组真核表达质粒，通过尾静脉高压注射方法证明该质粒确实有诱导小鼠体内DCs增多的功能，并且给小鼠注射质粒诱导DCs的效果与注射蛋白的效果接近。提示P333-FLT3L—hIgG质粒可代替FLT3L蛋白进行实验，为生产用于小鼠体内研究的FL33L蛋白提供了指导。
Objective Construct PTT3-FLT3L-hIgG Fc plasmid DNA, detect its protein expression levels in peripheral blood, identify the proportion and number of dendritic cells （DCs） in peripheral lymphoid organs, and evaluate the feasibility of hydrodynamic delivery instead of normal injection of protein. Methods FLT3L gene was amplified by PCR from P815 mast cell lines, and was inserted into PTT3-hIgG Fc vector. The PTT3-FLT3L-hIgG Fc plasmid was analysed by enzyme digestion and sequencing. Lymph node and spleen were harvested 9 days after hydrodynamic tail vein injection of PTT3-FLT3L-hlgG Fc and PTT3-blgG Fc plasmids. The proportion and number of DCs were identified by flow cytometry. Results The PTF3-FLT3L-bIgG Fc plasmid was proven correctly by enzyme digestion and sequencing. High levels of FLT3L-hIgG Fc protein can be detected in peripheral blood by ELISA after hydrodynamic tail vein injection. Besides, spleen was larger in mouse and CD11c^＋ MHC- II ^＋ DCs showed significant increase in peripheral lymphoid organs. Conclusion We constructed PTT3-FLT3L-hIgG Fc plasmid successfully and demonstrated that hydrodynamic tail vein injection of PTT3-FLT3L-hIgG Fc plasmids can increase DCs numbers greatly. What＇ s more, the effect of hydrodynamic delivery was comparable to injection of protein reported before. It indicated that we can use hydrodynamic delivery approach to achieve almost the same effect in experiments. The results further provided a foundation to produce FLT3L-hlgG Fc protein.
出处
《国际免疫学杂志》
CAS
2015年第2期99-104,共6页
International Journal of Immunology
基金
国家自然科学基金青年科学基金（31300728）
