Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay.
Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay.
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重组金鱼催乳素的制备及其在放射受体结合分析和放射免疫分析中的应用。
DOI：
10.1006/gcen.2001.7771
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发表时间：
2002
影响因子：
2.7
通讯作者：
C. H. Cheng
中科院分区：
文献类型：
--
作者：
A. Wong;Hilda Y. S. Cheung;E. K. Lee;K. Chan;C. H. Cheng
将金鱼催乳素cDNA亚克隆到pRSET A载体中，并在大肠杆菌中表达.重组金鱼催乳素主要以不溶性包涵体的形式表达，N-末端6x His-标记的融合蛋白。该融合蛋白经纯化、复性和（125）I标记，制备了用于受体结合的放射性配体，并验证了金鱼催乳素的放射免疫分析。使用金鱼鳃膜作为底物的催乳素受体结合，重组和天然形式的金鱼催乳素是有效的取代放射性配体的特异性结合在一个类似的剂量范围内，这表明融合蛋白被正确地重折叠，并可以识别金鱼催乳素受体。为了定量测定金鱼生物样品中催乳素的含量，建立了以（125）I标记的重组催乳素为示踪剂的放射免疫分析法。该测定显示对金鱼催乳素具有选择性，而与来自其他鱼类物种的哺乳动物催乳素和垂体激素（例如，生长激素和促性腺激素II）。这个新验证的测定系统被用来调查神经内分泌和信号转导机制调节催乳素释放的金鱼。在这种情况下，Ca（2+）离子载体A23187和蛋白激酶C激活剂TPA是有效的升高基础水平的催乳素分泌灌注金鱼垂体细胞。在平行的研究中，使用静态孵育的方法，生长抑素和多巴胺，但不是血管活性肠肽，抑制基础催乳素释放金鱼垂体细胞。这些结果表明，生长抑素和多巴胺可能是基础催乳素分泌的负调节因子，细胞外Ca（2+）内流和蛋白激酶C激活可能是介导金鱼催乳素释放的重要信号事件。
Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded, and (125)I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the (125)I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca(2+) ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca(2+) influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish.
蛋白激酶 C 的激活增加 GH3 垂体细胞分泌反应的 Ca2+ 敏感性。
DOI：
10.1152/ajpcell.1993.264.4.c1020
发表时间：
1993
期刊：
The American journal of physiology
影响因子：
--
作者：
Haymes,AA;Hinkle,PM
通讯作者：
Hinkle,PM
降钙素是去势雌性大鼠催乳素分泌的生理抑制剂。
影响因子：
4.8
作者：
Shah,GV;Pedchenko,V;Stanley,S;Li,Z;Samson,WK
通讯作者：
Samson,WK