Establishment of Functional Analysis by Expressing Antibody Molecule in the Cells

细胞内表达抗体分子功能分析的建立

基本信息

批准号：
07557151
负责人：
NAGAO Taku
金额：
$ 3.84万
依托单位：
The University of Tokyo (Granduate School of Pharm.Sci.)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

To analyze the functional differences among six G protein-coupled receptor kinases (GRKs) in the cells, we tried to establish a novel method, i.e.intracellular immunization. The intracellular immunization is a functional inactivation method by expressing monoclonal antibody (mAb). At first, mAb that reacts with GRK2 (betaARK1) was made by immunizing gluthatione-S-transferase (GST) fusion protein with carboxyl terminus of betaARK1. The resulting mAb specifically recognized betaARKl but not GRK3,5 and 6 with Western blot. The mAb was purified from culture supenatant of hybridoma by ammonium sulfate precipitation and Protein G column. We found that the purified mAb inhibited the basal and agonist-stimulated phosphorylating activities of betaARK1. The mAb also inhibited thc betagammabinding of heterotrimeric G protein to carboxyl terminus of betaARK1. As the GST-carboxyl terminus fusion protein could stimulate the phosphorylating activity of betaARKl, we concluded that antibody bound to the part to be essential for the activity and inhibited the activation of betaARKl. We have also injected mAb into myocytes and found the involvement of betaARKl in the process of beta _1 -adrenergic receptor-mediated modulation of L-type Ca ^<2+> channel. To construct single chain Fv molecule (scFv) to express mAb gene in the cell, the variable regions of heavy and light chains of mAb was amplified from mRNA of hybridoma by PCR.The scFv was expressed in E.coli. and the expressed scFv could recognize the betaARKl. We also ligated scFv into a cosmid vector to make a recombinant adenovirus. We believe that adenovirus-mediated expression of scFv can help to elucidate the roles of betaARKl in desensitization of various G protein-coupled receptors.

为了分析细胞中六个G蛋白偶联受体激酶（GRKS）之间的功能差异，我们试图建立一种新方法，即感染细胞免疫。细胞内免疫是通过表达单克隆抗体（MAB）的功能灭活方法。最初，通过与betaark1的羧基末端免疫胶状-S-转移酶（GST）融合蛋白来制作与GRK2（Betaark1）反应的mAb。由此产生的mAB特异性地识别了betaarkl，但没有wester blot的betaarkl，但没有GRK3,5和6。通过硫酸铵沉淀和蛋白质G柱从杂交瘤的培养层培养物中纯化MAB。我们发现纯化的mAb抑制了betaark1的基底和激动剂刺激的磷酸化活性。 MAB还抑制了异三聚体G蛋白的THC betagammabinding to betaark1的羧基末端。由于GST-羧基末端融合蛋白可以刺激Betaarkl的磷酸化活性，因此我们得出结论，与该部位结合的抗体对于活性至关重要，并抑制了Betaarkl的激活。我们还向肌细胞注入了mAb，发现betaarkl参与了β_1-肾上腺素能受体介导的L型Ca ^<2+>通道的调节。为了构建单链FV分子（SCFV）以表达细胞中的mAb基因，通过PCR从杂交瘤的mRNA中扩增了MAB的重和轻链的可变区域。并且表达的SCFV可以认识到Betaarkl。我们还将SCFV连接到宇宙载体中，以制造重组腺病毒。我们认为，腺病毒介导的SCFV表达可以帮助阐明βARKL在各种G蛋白偶联受体脱敏中的作用。