小尾寒羊骚痒病单抗制备及免疫组化检测方法的初步建立

Establishment of the Method of Immunohistochemistry Assay for the Detection of Scrapie by Monoclonal Antibody

目的通过表达羊PrPc核心片段、单抗制备,最后建立免疫组织化学检测羊痒病方法。方法用PCR方法从小尾寒羊基因组DNA中扩增编码PrPc核心片段DNA,并克隆到硫氧还原蛋白融合表达载体pET-32a(+)上,转化至E.coliBL21,IPTG诱导融合表达。以纯化的重组小尾寒羊PrPc核心片段免疫PrPc基因敲除小鼠,利用淋巴细胞杂交瘤技术,制备羊核心片段单克隆抗体。结果通过上述方法,得到相对分子质量约为35kD的重组小尾寒羊PrPc核心片段,筛选出六株能稳定分泌针对小尾寒羊PrPc核心片段特异单抗的杂交瘤细胞株。免疫组化试验表明,其中4株可特异性识别羊痒病脑组织切片中耐PK酶消化的PrPsc,经5次重复试验表明:所制备单抗与对照单抗F89结果完全一致。结论利用原核表达,单抗制备等技术制备出PrP特异性单克隆抗体,用笔者自己研制的单抗作为核心试剂初步建立了羊痒病免疫组化检测方法。

[Objective] The method of immunohistochemistry assay for the detection of Scrapie by monoclonal antibody was established. [Method] Genomic DNA was isolated from the ovine whole blood. Using the polymerase chain reaction to amplify the core part of PrP from the ovine Genomic DNA. By using the recombinant DNA technology, the recombinant protein of core part of PrP was obtained. Then using standard methodology of myeloma cell fusion, monoclonal antibodies were obtained to detect Scrapie by immunohistochemistry. [ Result ] The recombinant protein of core part of ovine PrP was obtained and a panel of six hybridoma cell lines secreting specific antibodies to core part of PrP related to Scrapie were established from one fusion between myeloma Sp2/0 and spleen cells from mice immunized with the purified recombinant protein. Four hybridoma cell lines can be used in immunohistochemistry assay for the detection of Scrapie. [Conclusion] The recombinant core part of PrP and special Mabs was obtained by using recombinant DNA and myeloma cell fusion technology. So the special monoclonal antibody developed in authors university can be used to detect PrP^sc of Scrapie by immunohistochemistry.