日本七鳃鳗Lj-RGD3毒素肽的3种单RGD模体突变体的克隆表达及其抑制Lewis细胞活性研究  

Molecular cloning and activity of recombinant proteins,rLj-113,rLj-114 and rLj-115,mutants contain only one RGD motif respectively transformed from Lj-RGD3 protein

作　　者：郑媛媛[1] 肖蓉[1] 王继红[1] 李庆伟[1] 

机构地区：[1]辽宁师范大学生命科学学院,辽宁大连116081

出　　处：《辽宁师范大学学报（自然科学版）》2017年第2期239-244,共6页Journal of Liaoning Normal University:Natural Science Edition

基　　金：国家高技术研究发展计划(863计划)资助项目(2014AA093502);国家自然科学基金资助项目(30471975);全国海洋公益资助项目(201305016-5)

摘　　要：为了评价Lj-RGD3 3个不同位点RGD模体的存在是否对该蛋白功能具有必要性及影响程度,采用全序列合成的方法合成了3种基于Lj-RGD3的RGD缺失突变体基因,并成功对其进行了pET23b载体的构建及重组蛋白的表达和纯化.将这3种分别保留第1位、第2位、第3位RGD模体的重组蛋白各命名为rLj-113、rLj-114和rLj-115.比较了这3种重组蛋白对Lewis小鼠肺癌细胞增殖、迁移、侵袭的影响,并对其是否能引起Lewis肺癌细胞凋亡进行了测定.结果显示,rLj-113、rLj-114和rLj-115对Lewis的增殖半抑制浓度IC_(50)值分别为2.73,2.75,2.53μmol/L,并且以剂量依赖的方式抑制Lewis小鼠肺癌细胞的增殖;突变体蛋白还能抑制以bFGF(basic fibroblast growth factor)为趋化剂的Lewis小鼠肺癌细胞的迁移及侵袭.综上可知,rLj-RGD3蛋白的RGD模体的位置并不影响其在抗Lewis小鼠肺癌细胞的生物活性,而保留单RGD模体的突变体rLj-113、rLj-114、rLj-115仍然具有抑制Lewis小鼠肺癌细胞增殖、迁移、侵袭以及诱导Lewis细胞凋亡的生物学活性.To explore whether the three RGD motifs on different positions have different effects on these functions,we got three mutant genes named rLj-113,rLj-l14,rLj-115 through the method of full sequence synthesis based on the RGD3 genes. We successfully expressed and purified the mutant proteins. We comparied biological activity about integrins associated proliferation, migration,invasion of these three recombinant proteins on Lewis lung cancer cells. The results showed that the three mu- tants inhibited the proliferation of the Lewis cells in a dose-dependent manner, the ICs0 are 2. 73, 2.75,2.53 /lmol/L. They can also inhibit integrin assoeiated proliferation,migration and invasion on Lewis cells in a dose-dependent manner. In conclusion,the position of the RGD motif on Lj-RGD3 did not affect the biological activity on anti-tumor cells, rLj-113, rL]-114, rL]-ll5 mutants remained only one RGD motif had the activity on inhibiting Lewis cells.

关 键 词：日本七鳃鳗 RGD毒素蛋白 突变体 细胞黏附 迁移及侵袭 

分 类 号：Q789[生物学—分子生物学]