摘要
采用逆转录-聚合酶链式反应（RT-PCR）方法，从鲤鱼脑垂体获得了两种GtHβ亚基的cDNA，克隆在pMD18-T载体。经测序确证后，将这两个基因克隆到原核表达载体pET-32（a）中，转化E．coli表达菌BL21（DE3），以IFFG诱导融合蛋白的高效表达。利用初步纯化后的抗原免疫新西兰大白兔，制备多克隆抗体。应用制备的兔抗血清与抽提的鲤鱼脑垂体的总蛋白分别进行Westem-blot及ELISA分析，结果显示获得的多抗能特异识别各自的天然蛋白。该结果为纯化天然GtH蛋白提供了有效的检测手段，为进一步制备GtH单市降抗体奠定了基础。
The article gives a description of the cloning, expression and polyclonal antibody preparation of two different gonadotropin （GtH） beta subunits complementary DNAs （cDNAs） of the common carp （ Cyprinus carpio ）. By RT-PCR, the cDNAs encoding GtH Ⅰand GtH Ⅱ beta subunits were obtained from common carp pituitary and cloned into pMD18- T vector. Confirmed by sequencing, two right cDNAs were cloned into pET-32（a） vector and then transformed into E. coli BL （21） DE3 to constnlct two expression strains. The recombinant GtH Ⅰand GtHⅡ beta subunits were efficiently expressed in the form of fusion protein after induction with IFrG. After being primarily purified, the recombinant proteins were injected into New Zealand White rabbits to produce polyclonal antibodies. Western blot analysis showed that these produced polyclonal antibodies could bind not only to the expressed antigens, but also to the denatured ones extracted from common carp pituitary specifically. Furthermore, ELISA analysis demonstrated that these polyclonal antibodies could recognize the natural GtH Ⅰand GtHⅡ beta subunits respectively. The results laid a foundation for the detection and purification of natural GtH as well as their monoclonal antibody preparation.
出处
《高技术通讯》
CAS
CSCD
北大核心
2006年第6期633-637,共5页
Chinese High Technology Letters
基金
973计划（2001CB109006）和国家自然科学基金（30430540,30130540）资助项目.
关键词
鲤鱼
促性腺激素
基因克隆
原核表达
多克隆抗体
common carp, gonadotropin, gene cloning, prokaryotic expression, polyclonal antibody
