The recombinant expression plasmids pET-PrP-UPD was constructed by inserting Ure2p prion domain (UPD) gene into the recombinant vector pET-PrP, The target gene was successfully expressed in the host cell BL21 (DE3) when induced with IPTG. Specific reaction was found between expressed protein and monoclonal antibody 6H4 by Western blot assay. The purified PrP-UPD fusion protein was polymerized into amyloid fibrils, and exhibited partial resistance to proteinase K, Which similar to the structural characteristics of prion. The BALB/c mice were immunized with purified recombinant PrP-UPD fusion protein. Immunized splenocytes were collected and fused with SP2/0 myeloma cell line. Hybridoma culture supernatant was screened by ELISA. Three hybridoma cell lines designated 1G3, 3A4 and 4D1, secreting monoclonal antibodies (mAbs) against recombinant bovine prion protein, were obtained. 1G3 could secret mAbs against bovine cellar prion protein and its immunoglobulin subclasses were IgG2a. The ELISA ti-ters of the ascites fluid were 1:10 000. The specificity of the mAb 1G3 was confirmed with Western blot.%利用DNA重组技术将酵母Ure2p朊蛋白结构域(UPD)基因插入牛朊蛋白(BPrP)表达质粒pET-PrP中,构建原核表达载体pET-PrP-UPD.阳性质粒转化宿主菌BL21 (DE3),在IPTG诱导下获得高效表达.Westernblot检测表明表达的重组蛋白与单克隆抗体6H4呈现特异性反应,且纯化的PrP-UPD融合蛋白在体外具有聚集成淀粉样纤维、抵抗蛋白酶K消化等朊毒体的结构特点.利用纯化的PrP-UPD免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,经克隆和筛选,获得3株稳定分泌抗重组PrP单抗的杂交瘤细胞,分别命名为1G3、3A4、4D1,其中1G3分泌的单抗能识别细胞型牛朊蛋白,其Ig亚类为IgG2b,腹水ELISA效价为1×105,Western blot表明该单抗具有较强的特异性.
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