| Transmissible Spongiform Encephalopathies (TSES) are associated with pathological cellular prion protein, which called prion. Prion is unique in physical-chemical and biology properties compared with common pathogens, so far no nucleic has ever been found in prion. In this study, Function epitopes of the prion were analyzed through many technologies, such as gene cloning and expression, biology information method, real-time RT-PCR, histopathology, synthetic peptide, and antibody preparation, etc. Moreover, The biological characteristic of structure protein were probed into after analysis to function epitopes of the prion, which laid a solid foundation to further study the pathogenesis and detection method for TSE.1. PrP gene of seven species, which are bovine, yak, panda, rhesus, peacock, keet, was respectively cloned in this study. The PrP gene structure polymorphology and functional epitopes message of these species were obtained through molecular biological software in genetic evolution and homology analysis. On the basis, we acquired the PrPc function epitopes of structure protein and the comparative analysis of the bovine prion protein second structure.2. The synthetic antigen were designed, which was conjugated the keyhole limpet hemoyanin (KLH), according to the functional etitopes of bovine prion,and the functional etitopes was determined by goldenkey software. Using this antigen, rabbits were immunized and the antibody were acquired, and then immunity property was tested based on the specific immunity procedure. The results showed that antibody titer was as high as 1:10s. Through enzyme-linked immunosorbent assay (ELISA) and western blotting analysis, this antibody could react with both recombinant protein and purified bovine PrPc. The results demonstrated that antibody obtained in our study has a good immunity characteristic and prospective of developing test reagent.3. Structure protein of bovine prion was expressed successfully through prokaryotic expression system, and which might be recognized by its structure peptide antibody. The results described here showed that the highest yield of recombinant protein expression induced by IPTQ under 37C for 3h. The expression protein and peptide antibody has good reaction by western blotting analysis. The results indicated that we had successfully developed recombinant protein of bovine prion, which can be used for further structure biology study of prion.4. Six hamsters were injected in brain by purified prion recombinant structure protein (10 u g/ul, 3 ul), and two hamsters in control group were injected with phosphatic salt buffer. The animal was sacrificed by bleeding through eyeball vein in day 102, sampling from brain, cerebellum, obex. One parts of brain was fixed by formaldehyde, then prepared paraffin wax section, stained by HE stain and examined by microscope. The others were extracted total RNA and the difference of PrPc expressionwas tested using real-time RT-PCR in both the test and control groups. The results indicated that, within day 102, neither abnormal clinical symptoms was observed nor obviously difference between the test and control group, nor histopathological changes and apparently difference of PrP gene expression. The results of the study demonstrated that during the observer period, the recombinant protein failed to cause pathological change of test rat, and difference of PrP gene expression, which provided significantly basic information for prion structure biology based on the recombinant protein. |
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