兔脑γ-氨基丁酸结合蛋白(受体)在非洲爪蟾卵母细胞上的重建

作者采用盐酸胍法提取兔脑RNA,并经寡聚-(dT)-纤维素亲和层析分离获得完好的总mRNA,这种异源mRNA注入非洲爪蟾卵母细胞胞浆后经放射受体分析证实,γ-氨基丁酸结合蛋白(受体)能被重建于细胞膜上。该重建技术稳定可靠,为进一步进行γ-氨基丁酸受体的体外研究奠定了基础。

Reconstitution of Rabbit Brain GABA Binding Protein(Receptor)on Xenopus Oocytes

Gurdon's Xenopus laevis oocyte transla-tion system has been extensively employedto investigate the molecular biology ofgamma-aminobutyric acid (GABA) recep-tors in the brains of embryonic chick, rat,etc.As GABA and its receptor may playa role in the pathogenesis of hepaticencephalopathy and a rabbit model of ful-minant hepatitis has been established,wereconstituted the rabbit brain GABA bind-ing protein (receptor)on Xepopus laevisoocytes to offer a base for further studyof the pathological effects of GABAreceptor at molecular level. In this report, total RNA was extractedfrom rabbit brain by guanidine hydrochlo-ride method. The total RNA was then sub-Jected to oligo- (dT) --cellulose affinity chro-matography to isolate mRNA. All the RNAsamples achieved a high purity with anaverage OD_(260)/OD_(280) ratio of about 2. 10.The clear bands of 18 S and 28S rRNA inelectrophoresis implied that the RNA wasnot degraded and was therefore availablefor expression.After two days of incuba-tion of the oocytes injected with mRNA,radioreceptor assay indicated that satur-able and specifically displaceable GABAbinding sites were implanted onto theoocyte membrane. This result led us to theconclusion that rabbit brain GABA bindingprotein (receptor)can be reconstituted byinjecting exogenous mRNA inclusive ofthose encoding the receptor into Xenopusoocytes. However, the functional activityof the reconstituted receptor as a GABAgated chloride ion channel needs to befurther characterized by patch clamping.