朊蛋白N端和C端多肽特异性抗体的制备及ELISA检测技术的研究

Preparations of specific antibodies against PrP N-terminal or C-terminal peptides and preliminary study on establishment of ELISA diagnostic method

目的制备朊蛋白N端和C端多肽特异性抗体,并对ELISA方法在朊病毒病检测中的应用进行研究。方法构建人朊蛋白N端和C端多肽原核表达重组质粒,分别表达纯化融合蛋白。以此为抗原制备朊蛋白N端和C端多肽特异性抗体。ELISA和WesternBlot检测所制备抗体与重组和天然的PrP蛋白的免疫反应性。初步建立间接ELISA检测技术。结果所制备的N端和C端抗体可特异性识别重组全长PrP蛋白和相应的PrP片段,无明显交叉反应。C端抗体还可有效地识别感染羊瘙痒因子263K的仓鼠脑组织中经PK消化后的PrPSc,其WesternBlot反应带型与PrP单抗3F4相似。5000r/min离心处理脑组织悬液可有效保留上清中PrPSc成分而不影响ELISA检测。蛋白酶K虽经灭活处理,但可明显抑制重组和天然PrP在液相中与相应抗体的结合。间接ELISA方法可根据反应A值区分正常或感染动物样本。结论所制备的朊蛋白N端和C端抗体具有良好的特异性,C端抗体可用于实验性朊病毒病的检测。建立的间接ELISA方法可试用于朊病毒病的初步筛查。

Objective To prepare the specific antibodies against PrP N-and C-terminal segments individually based on previous works and to evaluate ELISA method in diagnosis of prion disease. Methods Prokaryotie expression plasmids containing human PrP N-or C-terminal sequence were constructed and the fusion proteins were expressed and purified from E. coli. The specific antibodies were elicited by immunization of the respective purified proteins into BALB/e mice. The immunoreaetivities of the prepared antisera with recombinant and native PrP proteins were evaluated by ELISA and Western Blot assays. An indirect ELISA method was established. Results The prepared PrP N-and C-segment specific antibodies recognized the full-length PrP23-231 and respective PrP segments, but not the opposite peptides. The C-segment antibody reacted with PrP^Se molecules in the brain tissues from serapie infected hamsters after digestion with PK, showing similar eleetropboresis patterns as monoelonal antibody 3F4 in Western Blot. Centrifuged at 5000 r/min maintained the PrP^Se fraction in the supernatant of brain homogenates, removing unspecific influence factor in further ELISA tests. However, presence of PK inhibited remarkably the binding activity of PrP with its antibody in liquid. The indirect ELISA could distinguish the samples from normal and infected animals, according to their A values. Conclusion The specificity of the prepared N-and C-segment specific antibodies was reliable. The C-segment antibody could be used in diagnosis of the experimental prion diseases and the established indirect ELISA could be tried in primary screening for prion diseases.