利用杆状病毒在昆虫细胞内表达斜带石斑鱼促性腺激素及其生物活性研究-生理学专业毕业论文.docxVIP

中山大学博士学位论文 中山大学博士学位论文 崔淼 GTH I、GTH IIl3、GTH Ip(5×10一，5×10。2年口5×10～rtg／bw·g))年ⅡhCG(O．1，1和10 Iu佑w·g)，18小时后尾静脉取血4。C离心用于性类固醇激素(T、E2)的测定，其 中取脑垂体、下丘脑和性腺组织用于测定GnRH、P450aromA、P450aromB、GTH I 13、GTH 1113和GTH0t mRNA水平。实验结果表明，注射重组蛋白后，能够促进斜 带石斑鱼血清中性类固醇激素(T、E2)分泌增加，同时P450aromA、P450aromB、 GTH IB、GTH 1113和GTHa mRNA表达水平升高，而GnRH mRNA表达水平下降。 其中重组斜带石斑鱼GTH II、GTH I、GTH 1113、GTH IB在促进性类固醇激素(T、 E2)分泌上随着浓度的增加而增强，在相同浓度条件下，GTH I表现出的作用效果 最好。 综上所述，本文利用杆状病毒在昆虫细胞内成功表达了斜带石斑鱼促性腺激 素并对其进行了生物活性研究。本文的研究结果证明重组促性腺激素具有生物活 性，可以促进斜带石斑鱼性类固醇激素的分泌以及引起基因表达水平的改变，并 可为以后深入研究斜带石斑鱼生长、繁育以及促性腺激素作用机制和内分泌调控 等方面提供物质基础。 关键词：斜带石斑鱼：促性腺激素；杆状病毒表达系统；昆虫细胞；生物活性 III 中山^学博士学位论文 中山^学博士学位论文 崔纛 Title：Production of Recombinant Orange—spotted Grouper (Epinephelus coioides)Gonadotropin Hormone in Insect Cells by Baculovirus Expression System and Their Biological Effect Major-Physiology Name：Cui Miao Supervisor：Lin Haoran(Academician) ABSTRACT Orange—spotted grouper，Epinephelus coioides，which belongs to Pereifomaes， Serranidae，Epinepheiitlae，Epinephetus，is a marine fish widely cultured in Sottthem China It is a11 important seawater breeding fishes．which is a protogynous hermaphroditic fish．Due to the insufficiency of studies on endocrine regulation mechanism of growth development and reproduction，some difficulties related to its propagation and growth was not solved in the artificial propagation．The cDNA sequence encoding orange—spoaed grouper gonadotropin hormone subunits were eoloned into baculovirus transfer vectors and expressed in insect SD cells and the recombinant gotmdotropin’s biological activity were studied by in vitro and in vivo bioassay． 1．The eDNA encoding orange-spotted grouper gonadotropin hormone(GTHa， GTH 113 and GTH H13)were cloned from pituitary total RNA with RT-PCR and single subunit transfer plasmid vector(pFB-HT-A-GTHⅡD，pFB—I—GTH【I成pFB-HT-A-GTH IB，pFB—l·GTH 113，pFB-H。rlA—GTHo，pFB一1一GTH叫and double transfer plasmid vector 0FB—GTH I and pFB-GTH II)were successfully constructed．The pFa