用3-马来酰亚胺基苯甲酸琥珀酰亚胺酯(MbS)法将人工合成的4种不同来源的促性腺激素释放激(gnrH)(即章鱼gnrH、海鞘gnrH-Ⅰ、七鳃鳗gnrH-Ⅰ和七鳃鳗gnrH-Ⅲ)分别与匙孔戚血蓝蛋白(klH)偶联后作为抗原,免疫新西兰大白兔获得gnrH抗体.用间接ElISA方法检测抗血清效价,并用WESTErn blOT鉴定其特异性.制备抗原肽亲和纯化柱,纯化多克隆抗体.结果显示,4种多克隆抗体效价高达1∶500 000(体积比),并能和相应的gnrH多肽特异性结合,纯化后的抗体在SdS-PAgE显示为单一条带.通过上述方法,获得4种高效价、高纯度的gnrH抗体,为低等动物gnrH的研究提供了理论依据.Four immunogens was prepared by conjugation of four synthetic GnRH peptides(octopus GnRH,tunicate GnRH-Ⅰ,lamprey GnRH-Ⅰand lamprey GnRH-Ⅲ) and carrier protein key-hole limpet hemocyanin(KLH) respectively by MBS method.Antibody production against each immunogen was initiated in vivo by emulsifying them in Freund′s adjuvant and administering them to 2-month-old male New Zealand rabbits by hypodermal injection.The titer of antiserums was tested by indirect ELISA,and the results showed that the titer of four antibodies was 1∶500 000.The results of Western blot analysis showed that each antibody could specifically bind to the corresponding GnRH peptide.Finally,antiserums were purified by affinity purification,and the purified antibody showed a single band in SDS-PAGE.These datas suggest that four GnRH antibody with high effect and high purity were produced,and they provide an useful tool for studying GnRH of lower animal.国家自然科学基金资助项目(41076081
