To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of h...To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.展开更多
B族Ⅰ型清道夫受体(scavenger receptor class B type I,SR-BI)是丙型肝炎病毒(hepatitis C virus,HCV)的受体之一,可以与HCV的包膜蛋白E2结合,介导病毒颗粒进入宿主细胞。伴侣分子PDZK1(PDZdomain containing 1)是一个含有4个PDZ结构...B族Ⅰ型清道夫受体(scavenger receptor class B type I,SR-BI)是丙型肝炎病毒(hepatitis C virus,HCV)的受体之一,可以与HCV的包膜蛋白E2结合,介导病毒颗粒进入宿主细胞。伴侣分子PDZK1(PDZdomain containing 1)是一个含有4个PDZ结构域的支架蛋白,其第一个PDZ结构域可以与SR-BI的C端结合,调节其稳定表达和正确定位。研究发现PDZK1基因敲除以后,HCVcc(cell culture produced HCVvirus)和HCVpp(HCV pseudotype particles)的感染性明显下降;重新转入PDZK1后,可以部分恢复感染性。研究表明PDZK1可促进HCV入侵并可能是通过与SR-BI的相互作用介导的。伴侣分子对受体分子的调节在HCV入侵中的作用可能成为HCV治疗的潜在靶标,有助于开发新的治疗方法。展开更多
糖尿病及心血管疾病等代谢相关疾病是全球化公共健康问题,2014年Lancet发文指出中国糖尿病患者人数已达9800多万,成为主要公众健康威胁之一[1]。据世界卫生组织(World Health Organization,WHO)报道[2],心血管疾病造成的死亡占每年全球...糖尿病及心血管疾病等代谢相关疾病是全球化公共健康问题,2014年Lancet发文指出中国糖尿病患者人数已达9800多万,成为主要公众健康威胁之一[1]。据世界卫生组织(World Health Organization,WHO)报道[2],心血管疾病造成的死亡占每年全球死亡人数的1/3,这些疾病带来了严峻的卫生经济负担。胆固醇是动物组织细胞不可缺少的重要物质,它不仅参与细胞膜的形成,也在代谢过程中起重要作用,与多种代谢性疾病的发生发展相关。展开更多
文摘To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.
文摘B族Ⅰ型清道夫受体(scavenger receptor class B type I,SR-BI)是丙型肝炎病毒(hepatitis C virus,HCV)的受体之一,可以与HCV的包膜蛋白E2结合,介导病毒颗粒进入宿主细胞。伴侣分子PDZK1(PDZdomain containing 1)是一个含有4个PDZ结构域的支架蛋白,其第一个PDZ结构域可以与SR-BI的C端结合,调节其稳定表达和正确定位。研究发现PDZK1基因敲除以后,HCVcc(cell culture produced HCVvirus)和HCVpp(HCV pseudotype particles)的感染性明显下降;重新转入PDZK1后,可以部分恢复感染性。研究表明PDZK1可促进HCV入侵并可能是通过与SR-BI的相互作用介导的。伴侣分子对受体分子的调节在HCV入侵中的作用可能成为HCV治疗的潜在靶标,有助于开发新的治疗方法。
文摘糖尿病及心血管疾病等代谢相关疾病是全球化公共健康问题,2014年Lancet发文指出中国糖尿病患者人数已达9800多万,成为主要公众健康威胁之一[1]。据世界卫生组织(World Health Organization,WHO)报道[2],心血管疾病造成的死亡占每年全球死亡人数的1/3,这些疾病带来了严峻的卫生经济负担。胆固醇是动物组织细胞不可缺少的重要物质,它不仅参与细胞膜的形成,也在代谢过程中起重要作用,与多种代谢性疾病的发生发展相关。
