1.Establishment of PrP(Sc) conversion based on serial PMCA in vitro.
Song SHI ; Chen-fang DONG ; Bao-yun ZHANG ; Qi SHI ; Gui-rong WANG ; Xin WANG ; Jun HAN ; Xiao-ping DONG
Chinese Journal of Virology 2008;24(4):282-286
In order to establish an amplification system in vitro with which the PrP(Sc) is able to convert PrP(C) into proteinase K-resistant isoform infinitely and whether this system is more efficient than conventional protein misfolding cyclic amplification (PMCA), scrapie strain 263K-infected hamster's brain homogenate and homologous normal brain homogenate were prepared, respectively. A new methodology, namely serial PMCA, was utilized to reveal the continuous propagation ability of PrP(Sc). Totally 8 rounds of serial PMCA were proceeded and each round contained 48 cycles of alternative sonication and incubation. Simultaneously 144 cycles of conventional PMCA was used as a control. The results showed the PrP(Sc) from hamsters' brain tissues of scrapie agent 263K could replicate efficiently and infinitely with serial PMCA compared with finite propagation of PrP(Sc) with conventional PMCA system. The study of PrP(Sc) continuous propagation in brain homogenate with serial PMCA may further provide insight into the unsettled mechanism of prions misfolding and replication and apply to detect trace amount of PrP(Sc).
Animals
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Brain
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metabolism
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Cricetinae
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PrPC Proteins
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chemistry
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PrPSc Proteins
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chemistry
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Protein Folding
2.Establishment of a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters infected with scrapie agent 263K.
Bao-yun ZHANG ; Chan TIAN ; Jun HAN ; Chen GAO ; Qi SHI ; Hui-ying JIANG ; Wei ZHOU ; Xiao-ping DONG
Chinese Journal of Experimental and Clinical Virology 2008;22(5):321-323
OBJECTIVETo establish a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters and to address the stability of the panel conserved under the specific condition, for evaluating the diagnostic techniques of human and animal's prion diseases.
METHODS30 brain tissues of hamsters infected with scrapie strain 263K intracerebrally and 30 ones of normal hamsters were enrolled in this panel. Each brain sample was prepared to 10%, 1% and 0.5% homogenates and aliquoted into stocks. The presences of PrP(Sc) in each brain sample were evaluated with PrP-specific Western Blots and partially with immunohistochemistry, and the stability of PrP(Sc) signals in each sample were repeatedly assessed half a year later and 3 years later.
RESULTSPrP(Sc) signals were detected in all stocks of 10% brain homogenates from infected hamsters, 26 out of 30 stocks of 1% homogenates and 19 out of 30 stocks of 0.5% homogenates. The assessments of PrP(Sc) signals in all samples half-year and three years later demonstrated almost unchanged. All homogenates of brain tissues of normal hamsters were PrP(Sc) negative.
CONCLUSIONA prion disease PrP(Sc) panel of the brain tissues, which includes 90 PrP(Sc) positive stocks and PrP(Sc) negative ones, was successfully established, with a reliable stability of PrP(Sc) signals.
Animals ; Brain ; metabolism ; Disease Models, Animal ; Humans ; Male ; PrPC Proteins ; pharmacokinetics ; PrPSc Proteins ; Prion Diseases ; metabolism ; Scrapie ; metabolism ; Tissue Distribution
3.Study on the correlation between membrane protein Flotillin-1 and PrPc endocytosis.
Ke REN ; Ke WANG ; Yin XU ; Long-Zhu LI ; Jin ZHANG ; Hui WANG ; Yu-E YAN ; Xiao-Ping DONG ; Chen GAO
Chinese Journal of Experimental and Clinical Virology 2012;26(6):435-438
OBJECTIVETo explore whether the membrane-associated protein Flotillin-1 has relationship with endocytosis of PrPc.
METHODSThe expression of Flotillin-1 in different cell lines was detected with the method of Western Blot; the interaction between Flotillin-1 and PrPc in Cells which were treated with copper ions was observed using immunoprecipitation method.
RESULTS(1) Flotillin-1 was widely expressed in many cell lines without significant difference in the amounts of expression level; (2) Only in the appearance of copper ions, the protein complexes of PrPc and Flotillin-1 can be detected with the method of IP, which were related to copper ions concentration and processing time.
CONCLUSIONThe membrane-associated protein Flotillin-1 has the relationship with the endocytosis of PrPc.
Cell Line ; Cell Membrane ; genetics ; metabolism ; Endocytosis ; Humans ; Membrane Proteins ; genetics ; metabolism ; PrPC Proteins ; genetics ; metabolism ; Protein Binding ; Protein Transport
4.Preparation of PrP-specific Polyclonal Antibody Immunization of -knockout Mice with Recombinant Human PrP Protein.
Xue Hua YANG ; Yue Zhang WU ; Kang XIAO ; Li Ping GAO ; Dong Dong CHEN ; Xiao Ping DONG ; Qi SHI
Biomedical and Environmental Sciences 2020;33(7):493-501
Objective:
The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.
Methods:
We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.
Results:
Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.
Conclusion
The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.
Animals
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Antibodies
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immunology
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Blotting, Western
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Fluorescent Antibody Technique
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Immunization
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Immunohistochemistry
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Mice
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Mice, Knockout
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PrPC Proteins
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immunology
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PrPSc Proteins
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immunology
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Prion Proteins
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immunology
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Recombinant Proteins
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immunology
5.Preparation of monoclonal antibodies against prion proteins with full-length hamster PrP.
Xin-Li XIAO ; Hui-Ying JIANG ; Jin ZHANG ; Jun HAN ; Kai NIE ; Xiao-Bo ZHOU ; Yin-Xia HUANG ; Lan CHEN ; Wei ZHOU ; Bao-Yun ZHANG ; Yong LIU ; Xiao-Ping DONG
Biomedical and Environmental Sciences 2005;18(4):273-280
OBJECTIVETo prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases.
METHODSSeveral 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.
RESULTSThe 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.
CONCLUSIONThe mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.
Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Brain ; metabolism ; Cell Line, Tumor ; Cricetinae ; Enzyme-Linked Immunosorbent Assay ; Female ; Immunization ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; PrPC Proteins ; genetics ; immunology ; PrPSc Proteins ; genetics ; immunology ; Recombinant Proteins ; immunology
6.Dynamic analyses of PrP and PrP(Sc) in brain tissues of golden hamsters infected with scrapie strain 263K revealed various PrP forms.
Jian-Mei GAO ; Chen GAO ; Jun HAN ; Xiao-Bo ZHOU ; Xin-Li XIAO ; Jin ZHANG ; Lan CHEN ; Bao-Yun ZHANG ; Tao HONG ; Xiao-Ping DONG
Biomedical and Environmental Sciences 2004;17(1):8-20
OBJECTIVETo expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrP(Sc) during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrP(Sc) forms.
METHODSHamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrP(Sc) were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayed by electron microscopy analysis (EM) and immuno-golden EM.
RESULTSPrP(Sc) was initially detected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrP(Sc) in brain tissues increased along with the incubation. Several high and low molecular masses of PrP were seen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions.
CONCLUSIONThese results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrP(Sc) may indwell in brain tissues during the infection.
Animals ; Blotting, Western ; Brain ; metabolism ; ultrastructure ; Cricetinae ; Disease Models, Animal ; Female ; Glycosylation ; Immunohistochemistry ; Mesocricetus ; Microscopy, Electron ; PrP 27-30 Protein ; analysis ; metabolism ; PrPC Proteins ; analysis ; metabolism ; PrPSc Proteins ; analysis ; metabolism ; Scrapie ; metabolism ; pathology
7.Stable inhibition of human prion protein through a retrovirus-based RNAi system.
Wenjing XU ; Di WANG ; Juan WANG ; Huaiyi YANG
Chinese Journal of Biotechnology 2009;25(7):1022-1027
Prion leads to fatal transmissible spongiform encephalopathies. Cellular prion protein (PrPc) is necessary in prion disease. At present, it is demonstrated that PrPc plays a protective role in several carcinomas, such as gastric and breast cancer. We designed four 19-nt siRNAs according to cDNA sequence of human PrPc and constructed retrovirus-based RNAi vectors. We evaluated the inhibitive effect of these sequences on HuPrPc (human PrPc) and selected out three sequences with stable and efficient inhibition. And the efficiency of si626 reached more than 85%, which effect was significant. Next, we performed cell invasion assays of PC3M-si292 and PC3M-si626 in which PrPc was inhibited. And it showed that the cell invasive ability decreased in PrPc knock-down cell lines. This will make preparations for the further research on gene therapy of prion diseases and PrPc related carcinoma treatment and PrPc could be considered as a potential therapeutic target molecule in prostate cancer treatment.
Base Sequence
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Cell Line, Tumor
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DNA, Complementary
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genetics
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Genetic Therapy
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Humans
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Male
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Molecular Sequence Data
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PrPC Proteins
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biosynthesis
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genetics
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Prostatic Neoplasms
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drug therapy
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RNA Interference
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RNA, Small Interfering
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genetics
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Retroviridae
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genetics