人血管内皮生长因子受体-2基因（KDR）在链霉菌和大肠杆菌的克隆表达及其抑制剂筛选模型的构建研究
摘要
血管生成是毛细血管从已构建的毛细血管网中以出芽方式新生的过程,参与许多病理和生理过程如伤口愈合、组织再生、子宫内膜周期性增生以及肿瘤的转移和血管生成。尤其在实体瘤的恶性生长和转移中,肿瘤的血管生成起到重要的作用。血管内皮生长因子(Vascular endothelial growth factor,VEGF)作为血管内皮细胞特异的有丝分裂原,直接参与了血管形成这个过程,促进内皮细胞分裂增殖,促使新生血管形成,同时还可有效地提高血管的通透性。VEGF这一功能是通过和其特异性受体结合来发挥作用的。VEGF受体家族主要包括三个成员:VEGFR-1(Flt-1)、VEGFR-2(KDR/Flk-1)、VEGFR-3(Fit-4),它们都属于受体酪氨酸激酶(RTK)Ⅲ型超家族。研究表明VEGFR-2(KDR/Flk-1)在调节内皮细胞增殖、分化反应中最为重要,是内皮细胞VEGF信号传导的主要执行者,VEGF与KDR结合使受体二聚体化,进一步激发KDR酪氨酸发生磷酸化,导致自身酶活性和下游信号相关酶类的激活,从而调节血管内皮细胞相关反应。新生血管的形成对恶性肿瘤的生长是必不可少的因素,因而以KDR为靶点进行药物设计和筛选可成为抗肿瘤血管生成的重要方略。
KDR基因全长5821bp,编码基因位于4q12,编码区为4068bp,编码1356个氨基酸。由胞外7个Ig样结构域,一个跨膜域和胞内域组成。胞外域由766个氨基酸组成,第2-3Ig样结构域是主要VEGF结合区。KDR胞内区有565个氨基酸组成,可分为激酶功能区、68个氨基酸的酪氨酸激酶插入区和羧基末端。
本研究从人脐静脉内皮细胞(HUVEC)中提取总RNA,以其为模板,通过RT-PCR扩增获得编码KDR编码胞外区1-3Ig样结构域(KDR-ED)和胞内酪氨酸激酶催化区域(KDR-CD)的基因片段。测序结果同已知KDR基因编码序列进行比较,结果一致。
以链霉菌为表达体系进行基因表达。利用本实验室自行构建的新型链霉菌—大肠杆菌穿梭质粒pSGLgpp作为KDR-ED和KDR-CD的基因表达载体。将KDR-ED和KDR-CD编码基因分别克隆至载体的gpp信号肽编码序列下游。重组质粒转化至S.lividans TK24原生质体,经再生得到转化子,重组菌分别命名为S.lividans[pSGLgpp/KDR-ED]和S.lividans[pSGLgpp/KDR-CD]。SDS-PAGE结果表明,S.lividans[pSGLgpp/KDR-CD]分泌表达44kD的特异蛋白条带。采用磷酸化的酪氨酸抗体在没有加入ATP的情况下对KDR-CD蛋白进行western blot检测,结果出现44kD的单一杂交带,表达的KDR-CD具有免疫学活性。S.lividans[pSGLgpp/KDR-ED]可能分泌表达37KD的重组KDR-ED。
此外在大肠杆菌中也分别进行了KDR-ED和KDR-CD编码基因的克隆表达。将KDR-CD基因克隆至载体pET-30a,获得重组质粒pET-30a/KDR-CD并转化至大肠杆菌BL21(DE3)。SDS-PAGE分析显示在41kD处有特异性蛋白条带出现,大部分以包涵体的形式存在。采用磷酸化的酪氨酸抗体进行Western blot检测,全菌及超声破碎的上清和包涵体均在41kD处有单一杂交带,说明表达的KDR-CD重组蛋白具有免疫活性。将KDR-ED基因分别采用载体pET-30a和载体pBV220,在大肠杆菌中均未获表达。
分别对链霉菌和大肠杆菌表达的重组KDR-CD蛋白进行纯化。S.lividans[pSGLgpp/KDR-CD]的培养上清液经70%饱和硫酸铵沉淀、SP SepharoseFF阳离子交换树脂层析和Q Sepharose FF阴离子交换树脂层析,获得KDR-CD纯品。E.coli[pET-30a/KDR-CD]产生的重组蛋白,经尿素溶解再进行复性处理后,进行Ni~(2+)—螯合亲和层析得到具有免疫活性的纯化重组蛋白。两种宿主表达的重组的KDR-CD纯化后进行SDS-PAGE分析,HPLC检测纯度均达到95%,且具有免疫活性。
采用ELISA方法研究纯化的KDR-CD蛋白的酪氨酸激酶活性,以poly(E4Y)为反应底物,对反应条件包括KDR-CD浓度、底物浓度、ATP及Mg~(2+)和Mn~(2+)的浓度进行了优化,结果表明两种体系表达的KDR-CD蛋白均具有酪氨酸激酶活性。
以纯化的KDR-CD蛋白为靶位,建立了KDR酪氨酸激酶的抑制剂筛选模型,已筛选了600余株微生物产物,经复筛获得了13株产生抑制物质的菌株,阳性率为2.17%。
本研究首次在链霉菌中成功分泌表达了KDR-CD的酪氨酸激酶催化域,并首次以原核系统表达的重组KDR蛋白构建了筛选酪氨酸激酶抑制剂的新型模型,具有较突出新颖性和可操作性,为从微生物来源获得抗肿瘤血管形成的新药奠定了基础。
Angiogenesis is defined as the development of new blood vessels from preexisting vessels,and it is a crucial process not only in normal physiology but also in pathological process such as embryonic development,wound healing,the normal menstrual cycle and tumor metastasis and tumor-associated blood vessels.Especially angiogenesis plays an important role in solid tumor growth and tumor metastasis. Vascular endothelial growth factor(VEGF) acts as a highly specific mitogen directly involving in the process of angiogenesis,which induces endothelial cells division and proliferation and also increases vascular permeability.VEGF performs its function through bindig to special receptors.Three high-affinity cognate endothelial receptors for VEGF have been identified:VEGFR-1/Flt-1,VEGFR-2/Flk-1/KDR,and VEGFR-3/Flt-4.These receptors belong to the subfamily of classⅢreceptor tyrosine kinases(RTKs).Studies have indicated that KDR is the major signal transducer for the differentiation and proliferation of endothelial cells.VEGF binding to KDR results in dimerization of receptor monomers,transphosphorylation by dimerized receptors and docking of signaling proteins to receptor phosphotyrosines.An increase in the intrinsic catalytic activity and creation of binding sites on the RTKs to recruit cytoplasmic signaling proteins are primary features of RTKs activation,resulting in endothelial cells-associated reaction.As angiogenesis is of crucial importance for tumor growth, effectively inhibiting KDR signaling is considered as an attractive target for drugs design and screening novel anticancer agents against tumor angiogenesis.
A full-length 5821bp KDR cDNA includes 4068bp-coding region encoding 1356 amino acids,whose encoding gene locates in 4q12.KDR is characterised by seven extracellular immunoglobulin(Ig)-like domains followed by a membrane-spanning region and a conserved intracellular tyrosine kinase domain.Extracellular region is composed of 766 amino acids,2-3 Ig-like domain are main binding sites for VEGF. Intracellular tyrosine kinase domain has 565 amino acids,consisting of a kinase domain split by an about 68-amino-acid kinase insert,and a carboxyl terminus.
With RNA extracted from human umbilical vein endothelial cell as the template, the KDR cDNA encoding 1-3 extracellular immunoglobulin(Ig)-like domains (KDR-ED) and the catalytic core in intracellular tyrosine kinase domain(KDR-CD) were amplified with RT-PCR.The sequence of amplified KDR-ED and KDR-CD was completely identical with the published data(GenBank Accession No.AF063658).
Using the shuttle plasmid(Streptomyces-E.coli) pSGLgpp as expression vector, KDR-ED and KDR-CD were cloned and expressed in S.lividans TK24 separately. After the gene KDR-ED and KDR-CD were cloned at the downstream of gpp signal peptide in the plasmid pSGLgpp separately,the recombinant plasmids were transformed into S.lividans TK24,and the strains were named as S.lividans[pSGLgpp/KDR-ED]and S.lividans[pSGLgpp/KDR-CD]respectively. SDS-PAGE showed that a special protein band with MW 44kD appeared on the gel using the expressed protein of S.lividans[pSGLgpp/KDR-CD].With an anti-phosphotyrosine antibody,the result of Western blotting of the recombiant KDR-CD identified a protein band of about 44 kD on the membrane.Such results demonstrated the recombiant KDR-CD expressed by S.lividans has immunonogical activity and the protein was already phosphated in Streptomyces. S.lividans[pSGLgpp/KDR-ED]could express recombinant KDR-ED with about MW 37kD.
At the meantime,with the plasmid as pET-30a as vector,the gene of KDR-ED and KDR-CD were cloned and expressed in E.coliBL21(DE3) seperately.With SDS-PAGE, the apparent molecular weight of expressed KDR-CD was about 41kD existing in inclusion body form mostly.With an anti-phosphotyrosine antibody,the result of western blotting showed that the observed protein band of about 41kD in whole pellet, the soluble fraction and the inclusion body,respectively.The results confirmed that the recombinant KDR-CD protein from E.coli has immunological activity and the protein was phosphated in E.coliBL21(DE3).With SDS-PAGE,the KDR-ED was not expressed in E.coli.
The recombinant KDR-CD expressed in E.coli and S.lividans were purified, respectively.The KDR-CD was purified from the supernant of S.lividans[pSGLgpp/KDR-ED]by the following steps:70%ammonium sulfate precipitation,cation exchange resin SP-sepharose Fast Flow and an anion exchange resin Q-sephorose Fast Flow.After the recombinant KDR-CD protein from E.coli[pET-3Oa/KDR-CD]was denatured in urea.Through renatured treatment and Ni-NTA affinity chromatograph,the purified KDR-CD protein was obtained.The result of SDS-PAGE showed that the recombinant KDR-CD,which was obtained from two kinds of prokaryotic hosts using different purification methods,was pure.HPLC analysis showed that the purify reached 95%,respectively.
Using ELISA method,tyrosine kinase activity of purified KDR-CD from two kinds of prokaryotic hosts was assayed,respectively.In these assays,poly(E4Y) was used as reacting substrate.The optimal condition was tested including the quantity of KDR-CD,the condition of substrate and ATP and the condition of Mg~(2+) and Mn~(2+). Results showed that KDR-CD from two kinds of prokaryotic hosts had tyrosine kinase avtivity.
With the purified KDR-CD as target,the screening model for tyrosine kinase inhibitors was constructed.More than 600 microbiological metabolites were screened. Among them,13 metabolites were found with the inhibiting activity and the positive rate was about 2.17%.
As showing above,KDR-CD was expressed secretly in Streptomyces,which has not reported before.With the recombinant KDR-CD from Streptomyces as target,a novel drug-screening model for inhibitors of tyrosine kinase has been established, which is more effective practice and creative.It will lay on good foundation for seeking novel drugs for anti-tumor angiogenisis.
KDR基因全长5821bp,编码基因位于4q12,编码区为4068bp,编码1356个氨基酸。由胞外7个Ig样结构域,一个跨膜域和胞内域组成。胞外域由766个氨基酸组成,第2-3Ig样结构域是主要VEGF结合区。KDR胞内区有565个氨基酸组成,可分为激酶功能区、68个氨基酸的酪氨酸激酶插入区和羧基末端。
本研究从人脐静脉内皮细胞(HUVEC)中提取总RNA,以其为模板,通过RT-PCR扩增获得编码KDR编码胞外区1-3Ig样结构域(KDR-ED)和胞内酪氨酸激酶催化区域(KDR-CD)的基因片段。测序结果同已知KDR基因编码序列进行比较,结果一致。
以链霉菌为表达体系进行基因表达。利用本实验室自行构建的新型链霉菌—大肠杆菌穿梭质粒pSGLgpp作为KDR-ED和KDR-CD的基因表达载体。将KDR-ED和KDR-CD编码基因分别克隆至载体的gpp信号肽编码序列下游。重组质粒转化至S.lividans TK24原生质体,经再生得到转化子,重组菌分别命名为S.lividans[pSGLgpp/KDR-ED]和S.lividans[pSGLgpp/KDR-CD]。SDS-PAGE结果表明,S.lividans[pSGLgpp/KDR-CD]分泌表达44kD的特异蛋白条带。采用磷酸化的酪氨酸抗体在没有加入ATP的情况下对KDR-CD蛋白进行western blot检测,结果出现44kD的单一杂交带,表达的KDR-CD具有免疫学活性。S.lividans[pSGLgpp/KDR-ED]可能分泌表达37KD的重组KDR-ED。
此外在大肠杆菌中也分别进行了KDR-ED和KDR-CD编码基因的克隆表达。将KDR-CD基因克隆至载体pET-30a,获得重组质粒pET-30a/KDR-CD并转化至大肠杆菌BL21(DE3)。SDS-PAGE分析显示在41kD处有特异性蛋白条带出现,大部分以包涵体的形式存在。采用磷酸化的酪氨酸抗体进行Western blot检测,全菌及超声破碎的上清和包涵体均在41kD处有单一杂交带,说明表达的KDR-CD重组蛋白具有免疫活性。将KDR-ED基因分别采用载体pET-30a和载体pBV220,在大肠杆菌中均未获表达。
分别对链霉菌和大肠杆菌表达的重组KDR-CD蛋白进行纯化。S.lividans[pSGLgpp/KDR-CD]的培养上清液经70%饱和硫酸铵沉淀、SP SepharoseFF阳离子交换树脂层析和Q Sepharose FF阴离子交换树脂层析,获得KDR-CD纯品。E.coli[pET-30a/KDR-CD]产生的重组蛋白,经尿素溶解再进行复性处理后,进行Ni~(2+)—螯合亲和层析得到具有免疫活性的纯化重组蛋白。两种宿主表达的重组的KDR-CD纯化后进行SDS-PAGE分析,HPLC检测纯度均达到95%,且具有免疫活性。
采用ELISA方法研究纯化的KDR-CD蛋白的酪氨酸激酶活性,以poly(E4Y)为反应底物,对反应条件包括KDR-CD浓度、底物浓度、ATP及Mg~(2+)和Mn~(2+)的浓度进行了优化,结果表明两种体系表达的KDR-CD蛋白均具有酪氨酸激酶活性。
以纯化的KDR-CD蛋白为靶位,建立了KDR酪氨酸激酶的抑制剂筛选模型,已筛选了600余株微生物产物,经复筛获得了13株产生抑制物质的菌株,阳性率为2.17%。
本研究首次在链霉菌中成功分泌表达了KDR-CD的酪氨酸激酶催化域,并首次以原核系统表达的重组KDR蛋白构建了筛选酪氨酸激酶抑制剂的新型模型,具有较突出新颖性和可操作性,为从微生物来源获得抗肿瘤血管形成的新药奠定了基础。
Angiogenesis is defined as the development of new blood vessels from preexisting vessels,and it is a crucial process not only in normal physiology but also in pathological process such as embryonic development,wound healing,the normal menstrual cycle and tumor metastasis and tumor-associated blood vessels.Especially angiogenesis plays an important role in solid tumor growth and tumor metastasis. Vascular endothelial growth factor(VEGF) acts as a highly specific mitogen directly involving in the process of angiogenesis,which induces endothelial cells division and proliferation and also increases vascular permeability.VEGF performs its function through bindig to special receptors.Three high-affinity cognate endothelial receptors for VEGF have been identified:VEGFR-1/Flt-1,VEGFR-2/Flk-1/KDR,and VEGFR-3/Flt-4.These receptors belong to the subfamily of classⅢreceptor tyrosine kinases(RTKs).Studies have indicated that KDR is the major signal transducer for the differentiation and proliferation of endothelial cells.VEGF binding to KDR results in dimerization of receptor monomers,transphosphorylation by dimerized receptors and docking of signaling proteins to receptor phosphotyrosines.An increase in the intrinsic catalytic activity and creation of binding sites on the RTKs to recruit cytoplasmic signaling proteins are primary features of RTKs activation,resulting in endothelial cells-associated reaction.As angiogenesis is of crucial importance for tumor growth, effectively inhibiting KDR signaling is considered as an attractive target for drugs design and screening novel anticancer agents against tumor angiogenesis.
A full-length 5821bp KDR cDNA includes 4068bp-coding region encoding 1356 amino acids,whose encoding gene locates in 4q12.KDR is characterised by seven extracellular immunoglobulin(Ig)-like domains followed by a membrane-spanning region and a conserved intracellular tyrosine kinase domain.Extracellular region is composed of 766 amino acids,2-3 Ig-like domain are main binding sites for VEGF. Intracellular tyrosine kinase domain has 565 amino acids,consisting of a kinase domain split by an about 68-amino-acid kinase insert,and a carboxyl terminus.
With RNA extracted from human umbilical vein endothelial cell as the template, the KDR cDNA encoding 1-3 extracellular immunoglobulin(Ig)-like domains (KDR-ED) and the catalytic core in intracellular tyrosine kinase domain(KDR-CD) were amplified with RT-PCR.The sequence of amplified KDR-ED and KDR-CD was completely identical with the published data(GenBank Accession No.AF063658).
Using the shuttle plasmid(Streptomyces-E.coli) pSGLgpp as expression vector, KDR-ED and KDR-CD were cloned and expressed in S.lividans TK24 separately. After the gene KDR-ED and KDR-CD were cloned at the downstream of gpp signal peptide in the plasmid pSGLgpp separately,the recombinant plasmids were transformed into S.lividans TK24,and the strains were named as S.lividans[pSGLgpp/KDR-ED]and S.lividans[pSGLgpp/KDR-CD]respectively. SDS-PAGE showed that a special protein band with MW 44kD appeared on the gel using the expressed protein of S.lividans[pSGLgpp/KDR-CD].With an anti-phosphotyrosine antibody,the result of Western blotting of the recombiant KDR-CD identified a protein band of about 44 kD on the membrane.Such results demonstrated the recombiant KDR-CD expressed by S.lividans has immunonogical activity and the protein was already phosphated in Streptomyces. S.lividans[pSGLgpp/KDR-ED]could express recombinant KDR-ED with about MW 37kD.
At the meantime,with the plasmid as pET-30a as vector,the gene of KDR-ED and KDR-CD were cloned and expressed in E.coliBL21(DE3) seperately.With SDS-PAGE, the apparent molecular weight of expressed KDR-CD was about 41kD existing in inclusion body form mostly.With an anti-phosphotyrosine antibody,the result of western blotting showed that the observed protein band of about 41kD in whole pellet, the soluble fraction and the inclusion body,respectively.The results confirmed that the recombinant KDR-CD protein from E.coli has immunological activity and the protein was phosphated in E.coliBL21(DE3).With SDS-PAGE,the KDR-ED was not expressed in E.coli.
The recombinant KDR-CD expressed in E.coli and S.lividans were purified, respectively.The KDR-CD was purified from the supernant of S.lividans[pSGLgpp/KDR-ED]by the following steps:70%ammonium sulfate precipitation,cation exchange resin SP-sepharose Fast Flow and an anion exchange resin Q-sephorose Fast Flow.After the recombinant KDR-CD protein from E.coli[pET-3Oa/KDR-CD]was denatured in urea.Through renatured treatment and Ni-NTA affinity chromatograph,the purified KDR-CD protein was obtained.The result of SDS-PAGE showed that the recombinant KDR-CD,which was obtained from two kinds of prokaryotic hosts using different purification methods,was pure.HPLC analysis showed that the purify reached 95%,respectively.
Using ELISA method,tyrosine kinase activity of purified KDR-CD from two kinds of prokaryotic hosts was assayed,respectively.In these assays,poly(E4Y) was used as reacting substrate.The optimal condition was tested including the quantity of KDR-CD,the condition of substrate and ATP and the condition of Mg~(2+) and Mn~(2+). Results showed that KDR-CD from two kinds of prokaryotic hosts had tyrosine kinase avtivity.
With the purified KDR-CD as target,the screening model for tyrosine kinase inhibitors was constructed.More than 600 microbiological metabolites were screened. Among them,13 metabolites were found with the inhibiting activity and the positive rate was about 2.17%.
As showing above,KDR-CD was expressed secretly in Streptomyces,which has not reported before.With the recombinant KDR-CD from Streptomyces as target,a novel drug-screening model for inhibitors of tyrosine kinase has been established, which is more effective practice and creative.It will lay on good foundation for seeking novel drugs for anti-tumor angiogenisis.
引文
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