人朊病毒多肽抗体的制备及应用  

Generation and application of prion peptide antibody to human prion

作　　者：孙宪锋[1] 董小平[1] 张宝云[1] 周伟[1] 王长安[1] 赵同兴[1] 洪涛[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《中华微生物学和免疫学杂志》2000年第1期70-73,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家863高科技发展计划资助项目!863102110305

摘　　要：目的　利用人工合成人朊病毒蛋白(PrP)多肽制备特异性抗PrP抗体,以期用于朊病毒相关疾病的临床诊断。方法　根据PrP基因编码的氨基酸序列合成多肽,与载体偶联后免疫动物,所制备的抗血清用ELISA及Westernblot鉴定。结果　ELISA检测表明所制备的抗血清可同多种PrP发生阳性反应;Westernblot结果显示抗血清可与正常组织PrPc和病理组织的PrPsc反应;用蛋白酶K处理,可鉴别二者。此外,人、小鼠和兔的脑组织细胞膜提取物在Westernblot中显示一相对分子质量约30×103的条带,而相应肝、脾细胞膜提取物中无此条带出现。结论　所制备的PrP多肽抗血清可识别正常的PrPc和病理的PrPsc,合成肽抗原是制备抗朊病毒PrP抗体的一条有效的途径,可用于临床病例的检测。Objective Establishment of an effective method for diagnosis of prion associated diseases. Methods An anti peptide antibody has been developed by immunizing rabbits with a peptide synthesized according to the amino acid sequences of human prion protein. The peptide was coupled with BSA and injected into rabbits subcutaneously. The sera were collected and the antibody activity was evaluated with ELISA and Western blot. Results The titer of the antibody in ELISA was estimated as high as 1∶320?000. The antibody was also reacted with a prokaryotic expressed PrP GST fusion protein and a PrP truncated protein with a titer of 1∶32?000 and of 1∶8?000, respectively. Western blot assay of the brain extracts from the mice and rabbits showed a positive band at 30kD when incubated with the antisera, but a similar positive band was not observed in the membrane extracts derived from liver and spleen. The extracts of the brain tissues from a cadavar died of non CJD Chinese also showed same PrP c bands. To test whether the newly prepared antisera were able to reach with the PrP sc , we prepared the brain extract from a scrapie infested mouse. After being digested with proteinase K for 3?h at 37℃, the extract was loaded on a 10% SDS PAGE for Western blotting. A unique band appeared at 30kD when mixed with the antisera, whereas the controls were totally negative. Conclusion These antisera may possibly be applied in the diagnosis of prion associated diseases in clinic.

关 键 词：朊病毒 多肽 抗体 病毒 制备 

分 类 号：R392[医药卫生—免疫学]