定量测定黑鲷生长激素受体mRNA的液相杂交/RNase保护法

目的　建立液相杂交 核糖核酸酶保护测定 (RibonucleaseProtectionAssay,RPA)技术定量检测黑鲷(Sparusmacrocephalus)生长激素受体mRNA水平。方法　将黑鲷生长激素受体cDNA片断亚克隆至pGEM T载体 ,制备特异性的放射性反义RNA探针及正义RNA ,将反义RNA探针与正义RNA及样品总RNA进行液相杂交 ,用RNaseA和RNaseT1 降解杂交产物的单链RNA ,双链杂交体得到保护 ,然后检测杂交体的分子大小及放射强度。结果　检测出了黑鲷生长激素受体反义RNA探针与正义RNA及样品总RNA的特异性杂交片断 ,由此建立了定量测定黑鲷生长激素受体mRNA的液相杂交 RNase保护法 ,并采用该方法在黑鲷的多种组织中检测出了生长激素受体基因的表达 ,表达水平在肝脏中最高。该结果与我们曾采用生长激素放射受体分析法对黑鲷生长激素受体所研究的结果相吻合。结论　为进一步深入研究鱼类生长激素受体分子内分泌的调控理论提供了有力手段。

Validation of RNase Protection Assay for Black Seabream (Sparus macrocephalus ) Growth Hormone Receptor mRNA

Objective To develop a specific RNase Protection Assay to measure black seabream growth hormone (GH) receptor mRNA. Methods GH receptor cDNA fragment of black seabream was subcloned in pGEM|T vector. Then a specific antisense GH receptor RNA probe with radioactivity was prepared to hybridize with sense GH receptor RNA and total RNA from tissues of black seabream respectively. After hybridized solutions were treated with RnaseA/RNAseT1, the protected double strand hybridized RNAs could be detected by Autoradiogram and radioactivity counting. Results A specific RNase Protection Assay to measure black seabream GH receptor mRNA was developed. GH receptor mRNA were detected in brain, liver, kidney, muscle, gonad, gut and gill of black seabream, with highest level in liver. The results of GH receptor mRNA distribution were accorded with our previous results by GH Radioreceptor Assay in black seabream. Conclusion We can study the molecular mechanism of endocrine regulation by the specific RNase Protection Assay in black seabream GH receptor.