人成釉蛋白抗体的制备及组织表达特异性

Preparation of human ameloblastin antibody and the tissue expression specificity

目的:制备抗人成釉蛋白抗体,观察成釉蛋白在各组织中的表达。方法:实验于2002-03在解放军第四军医大学基础部生物化学与分子生物学教研室完成。以重组、纯化的成釉蛋白C端肽为抗原,混入完全/不完全弗氏佐剂,免疫新西兰大白兔,经5次免疫后,颈动脉取血,分离血清并用饱和硫酸铵纯化,制备兔抗人成釉蛋白多克隆抗体,用双向免疫扩散试验和ELISA检测抗体的效价。用WesternBlot检测人成釉蛋白的组织表达特异性。结果:①ELISA检测结果:表明兔抗人成釉蛋白多克隆抗体效价达到1∶10000。②WesternBlot显示:成釉蛋白在人牙胚组织总蛋白中有特异性表达,相对分子质量约为65000,在脑、心、肝、脾、肺、肾、胰腺、胸腺、骨骼肌等组织中未见表达条带。结论:制备了抗人成釉蛋白抗体,为研究成釉蛋白在人牙胚中的组织表达以及利用抗体纯化蛋白提供了基础,从蛋白水平证实成釉蛋白为牙胚组织特异性蛋白,并证实人牙胚组织中的成釉蛋白相对分子质量约为65000。

AIM： To prepare anti-human ameloblastin antibody, by which to study the tissue specificity of human ameloblastin. METHODS： The experiment was performed in the Basic Department of Biochemistry and Molecular Biology of Fourth Military Medical University in March 2002. The C telopeptide of recombinant purified ameloblastin was used as the antigen to mixed with complete/incomplete Freund＇s adjuvant. The blood sample was obtained from the carotid artery （CA） of immune New Zealand white rabbits after 5 times of immunity, and the serum was separated purified with saturated ammonium sulphate to establish rabbit-anti-human ameloblastin multiclonal antibody. The titer of antibody was detected by double immunodissfusion test and ELISA. The tissue expression specificity of human ameloblastin antibody was determined by Western Blot. RESULTS ： ① ELISA detection showed that the titer of the rabbit-anti-human ameloblastin multiclonal antibody was about 1：10 000. ② Westem Blot indicated that the ameloblastin was specifically expressed in the tooth germ with a relative molecular mass of 65 000, but no expression-strap was found in the brain, heart, liver, spleen, lung, kidney, pancreas, thymus or skeletal muscle of human. CONCLUSION： The anti-human ameloblastin antibody is successfully prepared, which provide a basis for the research on the tissue expression of ameloblastin in the tooth germ as well as the purification of protein by the antibody, and it is proved from the aspect of protein that the ameloblastin is the specific protein of tooth tissue, moreover, the relative molecular mass of ameloblastin in human tooth germ was about 65 000.