滨蟹（Carcinus maenas (L.)）色素扩散激素的定量、免疫亲和纯化及序列分析

Quantification, immunoaffinity purification and sequence analysis of a pigment-dispersing hormone of the shore crab, Carcinus maenas (L.).

Lohr J, Klein J, Webster S G, Dircksen H

Institute of Zoophysiology, University of Bonn, Germany.

Comp Biochem Physiol B. 1993 Apr;104(4):699-706. doi: 10.1016/0305-0491(93)90200-o.

Abstract

A sensitive sandwich-ELISA for pigment-dispersing hormone (PDH) with a detection limit of approximately 5 fmol per well has been developed using primary IgG and secondary biotinylated IgG fractions from an antiserum against beta-PDH and streptavidin-peroxidase conjugate for detection. 2. ELISA determinations in different parts of the central nervous system of the shore crab Carcinus maenas revealed maximum levels of 15.1 pmol immunoreactive PDH in the two eyestalk ganglia of one crab, 3.6 pmol in the sinus glands, 2.8 pmol in the brain and 0.8 pmol in the thoracic ganglia. 3. Carcinus PDH was purified from whole eyestalk ganglia and sinus glands by use of a simple two-step purification procedure consisting of immunoaffinity-prepurification on an anti-PDH IgG-protein A-Sepharose column and HPLC. 4. Automated gas-phase sequencing of the purified peptide and FAB-mass spectroscopy unambiguously revealed the sequence of Carcinus PDH as NSELINSILGLPKVMNDAamide (M(r) 1927.2 Da), which is identical to the beta-PDH of other brachyuran crustaceans.

摘要

利用抗β-色素分散激素（PDH）抗血清中的一级IgG和二级生物素化IgG组分以及链霉亲和素-过氧化物酶缀合物进行检测，开发出了一种灵敏的夹心酶联免疫吸附测定法（ELISA）来检测PDH，每孔的检测限约为5飞摩尔。2. 对滨蟹Carcinus maenas中枢神经系统不同部位进行的ELISA测定显示，一只蟹的两个眼柄神经节中免疫反应性PDH的最高水平为15.1皮摩尔，窦腺中为3.6皮摩尔，脑中为2.8皮摩尔，胸神经节中为0.8皮摩尔。3. 通过一个简单的两步纯化程序从整个眼柄神经节和窦腺中纯化Carcinus PDH，该程序包括在抗PDH IgG-蛋白A-琼脂糖柱上进行免疫亲和预纯化和高效液相色谱法。4. 对纯化肽进行自动气相测序和快原子轰击质谱分析明确显示，Carcinus PDH的序列为NSELINSILGLPKVMNDAamide（分子量1927.2道尔顿），与其他短尾类甲壳动物的β-PDH相同。