CAJ | 학술논문

目的构建重组真核表达质粒PTT3-FLT3L-hIgG Fc,鉴定其诱导机体表达蛋白FLT3L-hIgG Fc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P815细胞系中扩增出FLT3L基因片段,连接到PTT3-hIgG Fc载体上,得到重组真核表达质粒PTT3-FLT3L-hIgG Fc;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-hIgG Fc质粒和PTT3-hIgG Fc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-hIgG Fc质粒构建成功.小鼠尾静脉高压注射PTT3-FLT3L-hIgGFc后,血清中持续含有高于生理水平的FLT3L-hIgG Fc蛋白,小鼠脾脏体积明显增大,淋巴结和脾脏中CD11 c+MHC-Ⅱ+DC明显增多.结论成功构建了PTT3-FLT3L-hIgG Fc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PTT3-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FLT3L蛋白提供了指导.
목적 구건중조진핵표체질립PTT3-FLT3L-hIgG Fc,감정기유도궤체표체단백FLT3L-hIgG Fc적수평이급유도소서림파결비장수돌상세포(DCs)적공능,탐색체내주사질립대체주사단백유도DCs방법적가행성.방법 용PCR방법종소서비대세포P815세포계중확증출FLT3L기인편단,련접도PTT3-hIgG Fc재체상,득도중조진핵표체질립PTT3-FLT3L-hIgG Fc;통과매절감정화측서검측목적기인핵감산서렬;미정맥고압주사PTT3-FLT3L-hIgG Fc질립화PTT3-hIgG Fc대조질립,9d후취소서림파결화비장,류식세포술검측CD3、CD19、DX5、CD11c、MHC-Ⅱ등적표체래감정DCs적비례화수량.결과 매절화측서결과현시PTT3-FLT3L-hIgG Fc질립구건성공.소서미정맥고압주사PTT3-FLT3L-hIgGFc후,혈청중지속함유고우생리수평적FLT3L-hIgG Fc단백,소서비장체적명현증대,림파결화비장중CD11 c+MHC-Ⅱ+DC명현증다.결론 성공구건료PTT3-FLT3L-hIgG Fc중조진핵표체질립,통과미정맥고압주사방법증명해질립학실유유도소서체내DCs증다적공능,병차급소서주사질립유도DCs적효과여주사단백적효과접근.제시PTT3-FLT3L-hIgG질립가대체FLT3L단백진행실험,위생산용우소서체내연구적FLT3L단백제공료지도.
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-hIgG Fc and PTT3-hIgG Fc plasmids.The proportion and number of DCs were identified by flow cytometry.Results The PTT3-FLT3L-hIgG 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 CD11 c + 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-hIgG Fc protein.