重组斜带石斑鱼生长激素及其抗血清在放射免疫测定中的应用

Application of the recombinant growth hormone and its antibody in the development of radioimmunoassay in the orange-spotted grouper(Epinephelus coioides)

将斜带石斑鱼 (Epinepheluscoioides)生长激素成熟多肽cDNA序列克隆到质粒pRSET ,与 6x组氨酸等原核编码序列融合获得重组质粒pRGH6 ,转入大肠杆菌BL2 1(DE3) ,获得高效表达 ,表达量占细菌总蛋白的4 3%。免疫印迹证明表达产物为含斜带石斑鱼生长激素的融合蛋白 ,Ni2 +亲合层析柱纯化融合蛋白 ,以此为抗原免疫家兔制备特异性的抗血清。以纯化的重组生长激素和特异性的抗血清建立斜带石斑鱼生长激素的放射免疫测定法 ,该方法的灵敏度、特异性和重复性均达到测定血液生长激素的水平。研究了多巴胺的受体激动剂阿扑吗啡对静态孵育斜带石斑鱼脑垂体碎片释放生长激素的影响 ,结果表明 。

In order to determine the effects of apomorphine(APO) on growth hormone(GH) release from the pituitary of the orange spotted grouper( Epinephelus coioides ),We established a radioimmunoassay(RIA) by using recombinant growth hormone(rgGH) of the grouper and its specific antiserum.GH cDNA of orange spotted grouper was cloned into a prokaryotic expression vector pRSET and expressed in Escherichia coli BL21(DE3) cells in a form of fusion protein.Its yield was estimated to be 43% of the total bacterial proteins.The GH fusion protein was purified by Ni 2+ chelated affinity chromatography and the antiserum was prepared by using the purified protein as an antigen to immune a rabbit.The recombinant rgGH was iodinated with Iodine 125 in lactoperoxidase method.The results showed that the sensitivity of the RIA was 15 625 pg.Inter ?and intro assay coefficients of variation(CV) were 4 24% and 8 07% respectively.Recombinant carp GH and goldfish prolactin did not show cross reactivity with rgGH.Serial dilution curve of incubation medium samples was parallel to the standard curve of rgGH.The recombinant rgGH contained fusion partner in the N terminal of the protein,but it did not affect the sensitivity and precision of the RIA.According to the results,the established RIA can be used to detect GH in orange spotted grouper. The pituitary fragments of orange spotted grouper were incubated with APO,the D 1/D 2 dopamine receptor agonist,and the in vitro effects of APO on the GH release were detected by the established grouper GH RIA.10 -6 mol/LAPO obviously stimulated the GH release from the pituitary fragments after 12 hr and 24 hr of incubation.APO(10 -7 ,10 -6 mol/L) stimulated the GH release in a dose dependent manner after 12 hr of incubation.It reached 232 78±14 11 ng/ml and 324 15±79 2 ng/ml(mean± SE,n =3) respectively,and the control was 149 72±5 84 ng/ml(mean± SE,n =3).These results demonstrate that dopamine receptor D1/D2 agonist,APO,stimulates GH release at the pituitary levels in orange spotted grouper as in other teleosts and that dopamine receptor D1 might be present in the regulation of GH release in grouper.