Prokaryotic Expression Of Mammalian Prion And Dopple And Preparation Of Monoclonal Antibody Against Bovine Prion

Monoclonal antibodies (mAbs) against mature prion protein (mPrP) ofQinchuan yellow cattle were prepared and identified on the basis of genecloning and expression and hybridoma technology. Those mAbs wereexpected to work in the immunological diagnosis of mad cow disease.The ORFs of prion protein (PrP) gene and Dpl gene of Qinchuan cattle,native born sheep of Gansu Province and the Han man were acquired by PCRmethods. Then the recombinant expression plasmids containing maure PrP(mPrP) gene and mature Dpl (mDpl) gene were constructed. The three fusionproteins of mPrP and mDpl were overexpressed in the corresponding E. coliinduced by IPTG. Western Blot analysis with mAb SAF-70 as the detectingantibody showed that the three fusion mPrPs exhibitied a goodreactiongenicity and formed dimers in the cells. Analysis of proteinase Kdigestion demonstrated that these recombinant mPrP couldn't have thecharacteristics of Proteinase K- resistant of the scrapie prion protein (PrP^Sc).The Balb/C mice were immunized with the purified bovine mPrP. Spleencells from the immunized mouse were fused with SP2/0 cells, and hybridswere selected for their ability to generate antibody against mPrP by indirectELISA. Ascetic mAbs were purified by immunoaffinity chromatography andfurther identified their characteristics. Seven hybridomas were identified toconstantly secrete the antibody against bovine mPrP, and these mAbsbelonged to IgG2a subtype except for mAb N17(IgG2b). The titers of the 7mAbs ranged from 10⁴ to 10⁶ and their affinity constants ranged from 10⁹ to10¹². The 7 mAbs not only specifically recognized mPrP of yellow cattle butalso cross-reacted with mPrP of sheep and human being. However, thosemAbs didn't react with mDpl. mAb N15, N25, N59, N63 and N67 shared thesimilar epitope, while N17 and N64 recognized different epitopes located inthe carboxylic domain of mPrP according to epitope analysis. Westem Blot analysis also demonstrated that mAb N64 and N17 could react with PrP^C ofthe healthy yellow cattle. MAb N64 and N17 obtained in the experiment maybe worked as the key diagnostic reagents for the detection of mad cow diseaseand laid foundation for the development of the diagnostic kits.