草鱼促性腺激素基因原核表达及多克隆抗体的制备动物学专业论文.docxVIP

蛋白进行Westem 蛋白进行Westem blot鉴定，结果显示重组蛋白表达正确。利用制备型 SDS．PAGE纯化、回收的蛋白分别免疫新西兰大白兔，制备了抗GTHIx 和抗FSHp的两个具有较高效价的多克隆抗体。 本研究首次选用融合表达载体pGEX．4T-1作为草鱼促性腺激素 基因的原核表达工具，成功构建了重组GTH原核表达质粒，表达出草 鱼重组促性腺激素。为进一步阐明促性腺激素在硬骨鱼类生殖周期调 控中作用的分子机制、完善鱼类(特别是鲤科鱼类)的繁殖内分泌学 研究具有重要的理论意义，而且在实践中可以为草鱼的人工繁殖提供 一条新的催产途径，有重要的应用前景。 关键词：草囱__(Ctenopharyngodon idella)，促性腺激素，基因克隆，原 核表达，多克隆抗体 glycoprotein glycoprotein hormones composed of a common alpha subunit and a hormone-specific 13 subunit，which has its biological specificity．The hormonal activity is expressed only after these two subunits binding together．The reproductive cycles of teleosts are regulated by Hypothalamic—Pituitary-Gonad(HPG)axis，in wich the GtHs secreted by the pituitary play a crucial role，being the key regulators of controlling the gonad development and the final sex maturation． The cDNAs encoding GTHa、FSH[)and LHp subunits were obtained from grass carp(Ctenopharyngodon fdetta)pituitary by RT-PCR(reverse transcription polymerase chain reaction)．Sequencing analysis revealed that ORFs(open reading fragments)of GTHcx，FSHp， LHB subunits cDNA were 3 57bp，393bp and 44 1 bp，which encoding proteins of 1 1 8，1 30 and 1 46 amino acids，respectively． Three cDNA sequences of GTHs were inserted into PGEX-4T-1 vectors，using EcoR I and Xho I restriction sites．The recombinant III Key Key words：grass carp(Ctenopharyngodon idella)，gonadotropin,gene cloning，prokaryotic expression，polyclonal antibodies IV 缩略语表Amp 缩略语表 Amp 氨苄青霉素钠盐(ampicillin Na salt) Kan 卡那霉素(kanamycin) Bp 碱基对(base pair) cDNA 互补DNA(complementary DNA) DEPC 焦碳酸二乙酯(diethyl pyrocarbonate) kD 千道尔顿(kilodalton) mRNA 信使RNA(messenger RNA) PBS 磷酸盐缓冲溶液(phosphate-buffered saline) PCR 聚合酶链式反应(polymerase chain reaction) PMSF 苯甲基磺酰氟(phenylmethylsulfonyl fluoride) SDS 十二烷基磺酸钠(sodium dodecylsulfate) TBS Tris缓冲盐溶液(Tris-buffered saline) 嘣s 三羟甲基氨基甲烷(trihydroxymethyl aminomethane) IPTG 异丙基硫代．p．D．半乳糖苷(isopropylthio-一D—galactoside) Rr．PCR 逆转录．聚合酶链式反应(reverse transcription-polymerase chain) SDS．PAGE SDS聚丙烯酰胺凝胶电泳(SDS—pol