日本七鳃鳗RGD毒素肽Lj-RGD2的克隆、表达及其抗血管新生活性  

Clonging,Expression and Some Biological Functions of Lj-RGD2 Toxin Peptide from Lampetra japonica

作　　者：王继红[1,2] 刘洋[1,2] 吕莉[3] 宁淑香[1] 刘欣[1,2] 李庆伟[1,2] 

机构地区：[1]辽宁师范大学生命科学学院,大连116029 [2]辽宁省生物技术与分子药物研发重点实验室,大连116029 [3]大连医科大学药学院药理教研室,大连116044

出　　处：《中国生物化学与分子生物学报》2012年第3期254-260,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.30770297);辽宁省教育厅创新团队项目(No.2008T102);辽宁省科学技术计划项目(No.2010402009);大连市重大科技攻关项目(No.2009E11SF127;No.2010E13SF143)~~

摘　　要：日本七鳃鳗口腔腺分泌蛋白Lj-RGD2毒素肽分子质量为8 kD,是含有2个RGD(Arg-Gly-Asp)模体序列的多肽.为了探讨其是否具有RGD毒素蛋白的功能,对其基因进行了克隆和表达,并对其重组蛋白进行了抗血管新生活性研究.从日本七鳃鳗口腔腺中提取mRNA,以自行设计的引物进行RT-PCR扩增,以获得Lj-RGD2基因;将获取的目的基因与pET23b载体连接,经转化、克隆、阳性转化子的筛选鉴定;将含组氨酸标签的pET23b-Lj-RGD2转化BL21菌中诱导表达;经亲和层析纯化,获得可溶性重组rLj-RGD2蛋白,并对重组rLj-RGD2蛋白的抗血管新生功能进行了研究.结果显示,rLj-RGD2蛋白的IC50为1.77μmol/L,并以剂量依赖方式抑制人脐静脉内皮细胞ECV304的增殖;rLj-RGD2蛋白还能抑制ECV304细胞对人工基质膜Matrigel的黏附、以BFGF(basicfibroblast growth factor)为趋化剂的迁移及侵袭.鸡胚绒毛尿囊膜CAM(chick chorioallantoic membrane)血管新生实验结果表明,rLj-RGD2蛋白能以剂量依赖方式抑制BFGF诱导的CAM血管新生.由此可见,rLj-RGD2蛋白具有RGD毒素肽的活性特征,其有望成为一种抗血管新生基因工程新药.Lj-RGD2 is an 8.2 kD peptide with two RGD（Arg-Gly-Asp） motifs derived from the buccal gland of Lampetra japonica.We have cloned Lj-RGD2 to study whether the expressed recombinant protein maintained the function of antiangiogenic activities of RGD toxin.The Lj-RGD2 cDNA was obtained by RT-PCR,and the recombinant soluble Lj-RGD2（rLj-RGD2） was produced by E.coli,then purified by using His-affinity chromatography.The result showed that rLj-RGD2 dose-dependently inhibited the proliferation of ECV304 cells induced by basic fibroblast growth factor with an IC50 of 1.77 μmol/L.The migration of ECV304 cells was also inhibited when passing through the polycarbonate filters（pore size of 8.0 μm） or making an invasion into the Matrigel.In the angiogenesis tests,rLj-RGD2 suppressed the angiogenesis in chick CAM.Our data demonstrated that rLj-RGD2 was an additional functional member of RGD toxins and might be used as an anti-angiogenesis drug to be developed.

关 键 词：日本七鳃鳗 RGD毒素蛋白 细胞黏附 迁移及侵袭 血管新生 

分 类 号：Q71[生物学—分子生物学]