Flk1胞外1-3区基因的克隆及核酸疫苗的构建

目的 :克隆并构建小鼠 Flk1胞外 1 - 3区核酸疫苗 ,并检验疫苗引起特异性免疫反应的能力。方法 :RT- PCR克隆 Flk1胞外 1 - 3区 ,构建核酸疫苗 pc DNA3.1 ( + ) -Flk1 Domain1 - 3,脂质体转染 COS7细胞 ,western- bolt检测表达 ;疫苗免疫小鼠 ;以小鼠血管内皮细胞为靶细胞 ,以免疫的小鼠脾细胞作为效应细胞 ,采用标准 4h51Cr释放法计算 CTL特异性杀伤率。结果 :扩增出 1 2 5 2 bp片段。疫苗 DNA自动测序证明所获基因片段序列阅读框架正确。Western- blot检测转染疫苗的 COS7细胞显示细胞中有分子质量为 44k Da的蛋白表达 ;疫苗组与对照组比较 ,CTL杀伤率差异有显著性。结论 :pc DNA3.1 ( + ) Flk1 -Domain1 - 3核酸疫苗可有效的引起针对 Flk1的免疫反应。

Clone of Domain 1-3 of extracellular part of Flk1 and construction of the DNA Vaccine against it can raise specific immune response

Objective: To clone the Domain1-3 of the excellular parts flk-1 and constructe a DNA vaccine targeting this gene part and to test its ability to raise cellular immunity were tested. Methods: The total RNA of the mouse liver tissue was ext racted and RT-PCR were performed to clone the Domain 1-3 of the flk-1. Then t he cloned gene was ligased to the pcDNA3.1( ) to construct DNA vaccine. The vaccine was transfected to COS 7 by liposome and its expression was tested by western -bl otting. To test its effectiveness to raise immune response, mice were divided in to three groups:immune group, plasmid group and saline group, which were imm uned 3 times, once every two week by vaccine, plasmid and saline respectfully; t hen the standard 4 hour 51Cr releasing test was employed to detect specific CTL . Results: A 1252bp gene part was cloned through RT-PCR, which agreed with the l ength of Domain1-3 of flk-1. DNA sequencing shows that it is the right sequenc e of the Domain1-3. Western-blot shows that a 44kDa protein was expressed by CO S 7 tran sfected by the DNA vaccine. compared with the control group, this vaccine could stimulate potent specific CTL activity against flk-1. Conclusion:T he DNA vaccine against Domain1-3 of flk-1 was successfully constructed and cou ld stimulate potent specific immune response,which provided a basis for the furt her study of anti-angiogenesis intervention targeting Flk1.