Establishment of an assay for PrP(Sc) detection based on streptomycin precipitation / 病毒学报

To establish a new Western blotting assay for PrP(Sc) detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP(Sc) was recovered by centrifugation. The enrichment of PrP(Sc) by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP(Sc), forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP(Sc) was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP(Sc) in the specimens from central nerve system, or from peripheral organs or body fluids.

Preliminary analyses for influence of mutant PrPs with different number of octapeptide / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells.</p><p><b>METHODS AND RESULTS</b>Mutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type.</p><p><b>CONCLUSION</b>These data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.</p>

Generation and identification of phage engineering antibodies library against hamster prion protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Generation and Identification of Phage Engineering Antibodies Library against Hamster Prion Protein.</p><p><b>METHODS</b>Fab antibodies were identified and confirmed. BALB/c mice were immuned with PrP proteins. After the third immunization, the total RNA was extracted from the mice spleens. The genes of heavy Fd Fragments and light chain of antibodies amplified by a series of specific primers of human IgG Fab fragment were cloned into phagemid vector pComb3.</p><p><b>RESULTS</b>The combinatorial Fab library were constructed successfully and the cloning efficiencies both of light chain and Fd fragments were about near 10(6). The Fab library were panned by four cycles and screened with purified haPrP23-231 antigen on microtiter plates. 12 mAbs were isolated after four cycles of panning, five of which were sequenced and resulted sequence data were analyzed by alignment with GenBank immunoglobulin genes. Two strain of new heavy and light chain genes of Fab antibodies were identified and confirmed.</p><p><b>CONCLUSION</b>The research in this article will provide foundation for study of diagnosis and therapy of prion.</p>

Screening of Molecular Biomarkers Associated with Heart Failure Derived from Arrhythmogenic Right Ventricular Cardiomyopathy / 中国生物工程杂志

Objective:To sieve molecular biomarkers associated with heart failure derived from arrhythmogenic right ventricular cardiomyopathy (ARVC).Methods:The comparative gene microarray analysis using individual left ventricular myocardial samples from 5 patients with heart failure resulting from ARVC undergoing transplantation and 5 matched samples from normal adult heart were performed.The accuracy rate of the differentially expressed genes obtained by gene microarray was further verified by quantitative real time RT-PCR.Results:83 genes (from a total of 35000) that were differentially expressed in diseased hearts versus normal hearts were identified.Among them thirty-seven genes were up-regulated and forty-eight genes were down-regulated in ARVC hearts compared with the normal hearts.Changes of gene expressions were most prominently observed in those belonging to the "metabolism" category.Eighty percent of the selected 30 differentially expressed genes from microarray analysis were confirmed by quantitative real time RT-PCR.The highly expressed level of atrial natriuratic peptide (ANP) in ARVC hearts that was confirmed by quantitative real time RT-PCR and immunohistochemistry was reported.Conclusion:For the first time to our knowledge,the altered expressed genes in ARVC hearts compared with the matched normal hearts were identified.The results are the base to further study the molecular mechanism and identify diseased-specific molecular biomarkers in heart failure derived from ARVC.

Preparation of monoclonal antibodies against prion proteins with full-length hamster PrP / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases.</p><p><b>METHODS</b>Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry.</p><p><b>RESULTS</b>The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPsSc.</p><p><b>CONCLUSION</b>The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.</p>