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ABSTRACT: Prion diseases, or transmissible spongiform encephalopathies, are neurodegenerative diseases, which affect human and many species of animals with 100% fatality rate. The most accepted etiology for prion disease is 'prion', which arises from the conversion from cellular PrPC to the pathological PrPSc. This review discussed the characteristic structure of PrP, including PRNP gene, PrPC, PrPSc, PrP amyloid, and prion strains.
Acta Biochimica et Biophysica Sinica 03/2013; · 1.38 Impact Factor
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Yuelong Shu,
Chris Ka-Fai Li,
Zi Li,
Rongbao Gao,
Qian Liang,
Ye Zhang,
Libo Dong,
Jiangfang Zhou,
Jie Dong,
Dayan Wang, [......],
Ming Wang,
Tian Bai,
Dexin Li, Xiaoping Dong,
Hongjie Yu,
Weizhong Yang,
Yu Wang,
Zijian Feng,
Andrew J Mcmichael,
Xiao-Ning Xu
[show abstract]
[hide abstract]
ABSTRACT: The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, sug-gesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abun-dant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.
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YanLing Han,
Yuan Li,
Juan Song,
Ying Wang,
Qi Shi,
Cao Chen,
BaoYun Zhang,
Yan Guo,
ChaoPing Li,
Jun Han, XiaoPing Dong
[show abstract]
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ABSTRACT: To break immune tolerance to prion (PrP) proteins using DNA vaccines.
Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests.
Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response.
Prion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.
Biomedical and Environmental Sciences 10/2011; 24(5):523-9. · 1.35 Impact Factor
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Yuelong Shu,
Chris Ka-fai Li,
Zi Li,
Rongbao Gao,
Qian Liang,
Ye Zhang,
Libo Dong,
Jiangfang Zhou,
Jie Dong,
Dayan Wang, [......],
Ming Wang,
Tian Bai,
Dexin Li, Xiaoping Dong,
Hongjie Yu,
Weizhong Yang,
Yu Wang,
Zijian Feng,
Andrew J McMichael,
Xiao-Ning Xu
[show abstract]
[hide abstract]
ABSTRACT: The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.
The Journal of Infectious Diseases 03/2010; 201(8):1173-7. · 6.41 Impact Factor
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Yan Zhang,
Zhen Zhu,
Weizhong Yang,
Jun Ren,
Xiaojuan Tan,
Yu Wang,
Naiying Mao,
Songtao Xu,
Shuangli Zhu,
Aili Cui, [......],
Qun Li, Xiaoping Dong,
Jing Zhang,
Yueping Zhao,
Junfeng Wan,
Zijian Feng,
Junling Sun,
Shiwen Wang,
Dexin Li,
Wenbo Xu
[show abstract]
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ABSTRACT: Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, is normally mild but can have life-threatening manifestations. It can be caused by enteroviruses, particularly Coxsackieviruses and human enterovirus 71 (HEV71) with highly variable clinical manifestations. In the spring of 2008, a large, unprecedented HFMD outbreak in Fuyang city of Anhui province in the central part of southeastern China resulted in a high aggregation of fatal cases. In this study, epidemiologic and clinical investigations, laboratory testing, and genetic analyses were performed to identify the causal pathogen of the outbreak. Of the 6,049 cases reported between 1 March and 9 May of 2008, 3023 (50%) were hospitalized, 353 (5.8%) were severe and 22 (0.36%) were fatal. HEV71 was confirmed as the etiological pathogen of the outbreak. Phylogenetic analyses of entire VP1 capsid protein sequence of 45 Fuyang HEV71 isolates showed that they belong to C4a cluster of the C4 subgenotype. In addition, genetic recombinations were found in the 3D region (RNA-dependent RNA polymerase, a major component of the viral replication complex of the genome) between the Fuyang HEV71 strain and Coxsackievirus A16 (CV-A16), resulting in a recombination virus. In conclusion, an emerging recombinant HEV71 was responsible for the HFMD outbreak in Fuyang City of China, 2008.
Virology Journal 01/2010; 7:94. · 2.34 Impact Factor
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Xiaoli Li,
Chenfang Dong,
Song Shi,
Guirong Wang,
Yuan Li,
Xin Wang,
Qi Shi,
Chan Tian,
Ruimin Zhou,
Chen Gao, Xiaoping Dong
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ABSTRACT: Prion protein (PrP) is considered to associate with microtubule and its major component, tubulin. In the present study, octarepeat region of PrP (PrP51-91) was expressed in prokaryotic-expressing system. Using GST pull-down assay and co-immunoprecipitation, the molecular interaction between PrP51-91 and tubulin was observed. Our data also demonstrated that PrP51-91 could efficiently stimulate microtubule assembly in vitro, indicating a potential effect of PrP on microtubule dynamics. Moreover, PrP51-91 was confirmed to be able to antagonize Cu(2+)-induced microtubule-disrupting activity in vivo, partially protecting against Cu(2+) intoxication to culture cells and stabilize cellular microtubule structure. The association of the octarepeat region of PrP with tubulin may further provide insight into the biological function of PrP in the neurons.
Acta Biochimica et Biophysica Sinica 11/2009; 41(11):929-37. · 1.38 Impact Factor
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Ping Li,
Chenfang Dong,
Yanjun Lei,
Bing Shan,
Xinli Xiao,
Huiying Jiang,
Xin Wang,
Chen Gao,
Qi Shi,
Kun Xu,
Chan Tian,
Jun Han, Xiaoping Dong
[show abstract]
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ABSTRACT: Doppel (Dpl) is a prion (PrP)-like protein due to the structural and biochemical similarities; however, the natural functions of Dpl and PrP remain unclear. In this study, a 531-bp human PRND gene sequence encoding Dpl protein was amplified from human peripheral blood leucocytes. Full-length and various truncated human Dpl and PrP proteins were expressed and purified from Escherichia coli. Supplement of the full-length Dpl onto human neuroblastoma cell SH-SY5Y induced remarkable cytotoxicity, and the region responsible for its cytotoxicity was mapped at the middle segment of Dpl [amino acids (aa) 81-122]. Interestingly, Dpl-induced cytotoxicity was antagonized by the presence of fulllength wild-type PrP. Analysis on fragments of PrP mutants showed that the N-terminal fragment (aa 23- 90) of PrP was responsible for the protective activity. A truncated PrP (PrPdelta32-121) with similar secondary structure as Dpl induced Dpl-like cytotoxicity on SHSY5Y cells. Furthermore, binding of copper ion could enhance the antagonizing effect of PrP on Dpl-induced cytotoxicity. Apoptosis assays revealed that cytotoxicity induced by Dpl occurred through an apoptotic mechanism. These results suggested that the function of Dpl is antagonistic to PrP rather than synergistic.
Acta Biochimica et Biophysica Sinica 02/2009; 41(1):42-53. · 1.38 Impact Factor
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Jianming Chen,
Chen Gao,
Qi Shi,
Guirong Wang,
Yanjun Lei,
Bing Shan,
Baoyun Zhang,
Chenfang Dong,
Song Shi,
Xin Wang,
Chan Tian,
Jun Han, Xiaoping Dong
[show abstract]
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ABSTRACT: The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein to pathologic isoform. Casein kinase II (CK2) is a ubiquitously expressed and evolutionarily conserved pleiotropic protein kinase that is essential for viability. To explore the possible molecular interaction between CK2 and prion protein (PrP), the full-length sequences of human CK2alpha and CK2beta complementary DNA were amplified with reverse transcription-polymerase chain reaction using the total messenger RNA from cell line SH-SY5Y as the template; then, the fusion proteins histidine-CK2alpha and glutathione S-transferase-histidine-CK2beta were expressed in Escherichia coli. The interaction between CK2 and PrP was evaluated with co-immunoprecipitation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2alpha, but not with CK2beta. The native CK2 and PrP in hamster brains interacted with each other, forming protein complexes. Three different glycosylated forms of PrP (diglycosylated, monoglycosylated and unglycosylated PrP) from normal brains interacted with the CK2alpha subunit, though the unglycosylated PrP seemed to have a stronger binding ability with CK2alpha subunit. The domain responsible for interacting with CK2alpha was located at the C-terminal segment of PrP (residues 91-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2alpha (both in recombinant and native categories), scientific clues for further assessing the potential biological significance of the PrP-CK2 interaction, and the possible role of CK2 in the pathogenesis of prion diseases.
Acta Biochimica et Biophysica Sinica 01/2009; 40(12):1039-47. · 1.38 Impact Factor
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Qi Shi,
Chen Gao,
Wei Zhou,
Bao-Yun Zhang,
Jian-Ming Chen,
Chan Tian,
Hui-Ying Jiang,
Jun Han,
Ni-Juan Xiang,
Xiao-Fang Wang,
Yong-Jun Gao, Xiao-Ping Dong
[show abstract]
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ABSTRACT: Human transmissible spongiform encephalopathies (HTSE), or Creutzfeldt-Jakob disease (CJD), is a group of rare and fatal diseases in central nervous system. Since outbreak of bovine spongiform encephalopathy (BSE) and variant CJD, a worldwide CJD surveillance network has been established under the proposition of WHO. In China, a national CJD surveillance system has started since 2002. The data of CJD surveillance from 2006 to 2007 was analyzed.
Total 12 provinces are included in CJD surveillance system. The surveillance unit in each province consists of one or two sentinel hospitals and the provincial CDC. All suspected CJD cases reported from CJD surveillance were diagnosed and subtyped based on the diagnostic criteria for CJD issued by WHO.
Total 192 suspected CJD cases were reported and 5 genetic CJD, 51 probable and 30 possible sporadic CJD (sCJD) cases were diagnosed. The collected sCJD cases distribute sporadically without geographical clustering and seasonal relativity and the highest incidences in both probable and possible sCJD cases appeared in the group of 60-69 year. The most common three foremost symptoms were progressive dementia, cerebellum and mental-related symptoms. The probable sCJD patients owning both typical EEG alteration and CSF protein 14-3-3 positive have more characteristic clinical syndromes than the ones having only one positive. The polymorphisms of codon 129 of all tested reported cases shows typical patterns of Han Chinese as previous reports, that M129M are predominant whereas M129V are seldom.
Chinese CJD patients possessed similar epidemiological and clinical characteristics as worldwide.
BMC Public Health 11/2008; 8:360. · 2.00 Impact Factor
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ABSTRACT: The pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with the generation of inflammatory and neuropathic pain. The current study aims to investigate the expression of TNF-α in the brain of rats with spared nerve injury (SNI), a neuropathic pain model with the lesion of common peroneal and tibial nerves. Two weeks following SNI, the immunohistochemical results identified that the expression level of TNF-α in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the roles of TNF-α in the development of neuropathic pain, different doses of anti-TNF-α antibody (20, 2.0 and 0.2 μg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The results showed that the 50% paw withdrawal threshold (von Frey test) of SNI rats were increased by 20 and 2.0 μg/ml anti-TNF-α antibody as compared with that of the basic value and control groups (P<0.05), the analgesic effect lasted for 50 and 30 min, respectively. However, no significant analgesic effect was observed after 0.2 μg/ml antibody was microinjected into the RN. These results suggest that the TNF-α of RN is involved in the development of neuropathic allodynia in SNI rats.
Brain research bulletin 10/2008; 77(5):233-6. · 2.18 Impact Factor
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Run An,
Chenfang Dong,
Yanjun Lei,
Lu Han,
Ping Li,
Jianming Chen,
Guirong Wang,
Qi Shi,
Chen Gao,
Huiying Jiang,
Wei Zhou,
Jun Han,
Yonglie Chu, Xiaoping Dong
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ABSTRACT: One of the physiological functions of cellular prion protein (PrP(C)) is believed to work as a cellular resistance to oxidative stress, in which the octarepeats region within PrP plays an important role. However, the detailed mechanism is less clear. In this study, the expressing plasmids of wild-type PrP (PrP-PG5) and various PrP mutants containing 0 (PrP-PG0), 9 (PrP-PG9) and 12 (PrP-PG12) octarepeats were generated and PrP proteins were expressed both in E. coli and in mammalian cells. Protein aggregation and formation of carbonyl groups were clearly seen in the recombinant PrPs expressed from E. coli after treatment of H(2)O(2). MTT and trypan blue staining assays revealed that the cells expressing the mutated PrPs within octarepeats are less viable than the cells expressing wild-type PrP. Statistically significant high levels of intracellular free radicals and low levels of glutathione peroxidase were observed in the cells transfected with plasmids containing deleted or inserted octarepeats. Remarkably more productions of carbonyl groups were detected in the cells expressing PrPs with deleted and inserted octarepeats after exposing to H(2)O(2). Furthermore, cells expressing wild-type PrP showed stronger resistant activity to the challenge of H(2)O(2) at certain extent than the mutated PrPs and mock. These data provided the evidences that the octarepeats number within PrP is critical for maintaining its activity of antioxidation. Loss of its protective function against oxidative stress may be one of the possible pathways for the mutated PrPs to involve in the pathogenesis of familial Creutzfeldt-Jacob diseases.
Science in China Series C Life Sciences 08/2008; 51(7):630-9. · 1.61 Impact Factor
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Yuelong Shu,
Yu Lan,
Leying Wen,
Ye Zhang,
Jie Dong,
Xinsheng Zhao,
Dayan Wang,
Lihong Yao,
Xiyan Li,
Wei Wang, [......],
Jingjing Huang,
Lei Yang,
Hongjie Yu,
Yuanji Guo,
Weizhong Yang,
Xiyan Xu,
Nancy J. Cox, Xiaoping Dong,
Yu Wang,
Dexin Li
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ABSTRACT: Twenty cases of H5N1 influenza virus infection in humans that resulted in 13 deaths were identified in China between November 2005 and July 2006. In this study, we performed genetic and antigenic analyses on the 16 H5N1 virus strains isolated from these infected persons. Sequence data obtained in our study indicates that isolates from southern and northern China each form unique sublineages among H5N1 viruses, with the isolates from southern China clustering in a novel clade. Antibodies raised against the H5N1 virus A/Vietnam/1194/2004, a candidate for current human pandemic vaccine production and clinical trials, had very limited cross-reactivity with the Chinese H5N1 isolates. Our study contains valuable information for global pandemic vaccine selection by providing evidence for the need to develop vaccine candidates selected from the H5N1 viruses isolated from these more recent human cases from China.
05/2008: pages 103 - 112; , ISBN: 9780470770672
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Wei Wang,
Chen Wang,
Shuqing Xu,
Chen Chen,
Xiaomei Tong,
Yu Liang, Xiaoping Dong,
Yanjun Lei,
Xiaoguang Zheng,
Jun Yu,
Jian Wang
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ABSTRACT: Human papillomavirus-2 (HPV-2) is generally associated with common warts. The association of cutaneous horns with HPV-2 infection has never been reported.
To identify the papillomavirus (PV) type(s) involved in cutaneous horns and analysis the genomes of these viruses.
We screened biopsies from two patients with multiple cutaneous horns using PV type-specific PCR assays, and sequenced the whole genomes of the viruses by a PCR-by-PCR strategy. Sequence comparison with the reference genome and its closely related PVs in the same phylogenetic group was performed to identify sequence variation across the genome(s) of newly detected PV(s).
Two strains of HPV-2 were identified from the biopsies of two patients respectively. No double or multiple infections were detected. Novel mutations were found in the HPV-2 genome, located both in the coding and non-coding regions. Amino acid changes occurred only in E1 and E7 ORFs. The two strains also shared several mutations at the same positions.
Each patient was infected with a single strain of HPV-2 that developed unique mutations; HPV-2 may play a role in the onset and development of cutaneous horns; amino acid changes in functionally significant viral proteins may confer differential pathogenic risks.
Journal of Clinical Virology 06/2007; 39(1):34-42. · 3.97 Impact Factor
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Jun Han,
Jin Zhang,
Hailan Yao,
Xiaofan Wang,
Feng Li,
Lan Chen,
Chen Gao,
Jianmei Gao,
Kai Nie,
Wei Zhou, Xiaoping Dong
[show abstract]
[hide abstract]
ABSTRACT: Microtubule-associated protein tau is considered to play roles in many neurodegenerative diseases including some transmissible spongiform encephalopathies. To address the possible molecular linkage of prion protein (PrP) and tau, a GST-fusion segment of human tau covering the three-repeat region and various PrP segments was used in the tests of GST pull-down and immunoprecipitation. We found tau protein interacted with various style prion proteins such as native prion protein (PrPc) or protease-resistant isoform (PrPSc). Co-localization signals of tau and PrP were found in the CHO cell tranfected with both PrP and tau gene. The domain of interaction with tau was located at N-terminal of PrP (residues 23 to 91). The evidence of molecular interactions between PrP and tau protein highlights a potential role of tau in the biological function of PrP and the pathogenesis of TSEs.
Science in China Series C Life Sciences 11/2006; 49(5):473-9. · 1.61 Impact Factor
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Jiangtao Lin,
Jiansan Zhang, Xiaoping Dong,
Hanhua Fang,
Jiangting Chen,
Nan Su,
Qiang Gao,
Zhenshan Zhang,
Yuxuan Liu,
Zhihong Wang, [......],
Ruihua Sun,
Changgui Li,
Su Lin,
Mei Ji,
Yan Liu,
Xu Wang,
John Wood,
Zijian Feng,
Yu Wang,
Weidong Yin
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ABSTRACT: Avian influenza A virus H5N1 has caused widespread infections that have resulted in severe disease or death in poultry and wild birds as well as human beings. This virus has the potential to emerge as a pandemic threat and H5N1 vaccines are being developed in many countries. Our aim was to assess the safety and immunogenicity of an inactivated adjuvanted whole-virion H5N1 vaccine.
A stratified randomised, placebo-controlled, double-blind phase I clinical trial was done in 120 volunteers aged 18-60 years. Volunteers were assigned to receive two doses of placebo (n=24) or an inactivated whole-virion influenza A (H5N1) vaccine with 1.25 microg (24), 2.5 microg (24), 5 microg (24), or 10 microg (24) haemagglutinin per dose with aluminium hydroxide adjuvant on day 0 and 28. Serum samples were obtained on day 0, 14, 28, 42, and 56 for haemagglutination inhibition and virus neutralisation assays. This trial is registered with the ClinicalTrials.gov registry with the number NCT00356798.
All four formulations of vaccines were well tolerated. No serious adverse event was reported and most local and systemic reactions were mild and transient. All formulations induced antibody responses after the first dose; the highest immune response of 78% seropositivity was seen in the 10 mug group after two vaccine doses. Two individuals dropped out: one in the 1.25 microg group (withdrew consent) and one in the 10 microg group (discontinued); one individual was also excluded from the final analysis.
A two-dose regimen of an adjuvanted 10 microg inactivated whole-virion H5N1 vaccine met all European regulatory requirements for annual licensing of seasonal influenza vaccine. Lower doses of this vaccine could achieve immune responses equivalent to those elicited by adjuvanted or non-adjuvanted split-virion vaccines. The use of a whole virion vaccine could be more adaptable to the antigen-sparing strategy recommended by WHO for protection against an influenza pandemic.
The Lancet 10/2006; 368(9540):991-7. · 38.28 Impact Factor
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Jiansan Zhang,
Yuxuan Liu,
Liangxiang Hu,
Qiang Gao,
Zhenshan Zhang,
Xiaomei Zhang,
Jiangting Chen,
Xuejie Gong,
Lifei Song,
Yufen Liu,
Jing Li,
Shufen Li,
Jinfeng Huang,
Ye Ning,
Hong Gao,
Chuan Qin, Xiaoping Dong,
Jianguo Wei,
Guanmu Dong,
Weidong Yin
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ABSTRACT: A reference antiserum for SARS is in urgent need in the development of SARS vaccine and other serological test of SARS research. Convalescent serum was collected from clinical confirmed patient. ELISA, Western-blotting and neutralization assay detected specific antibody against SARS coronavirus. This antiserum was prepared as in-house reference antiserum, freeze-dried and sealed in ampoules. The potency of this reference antiserum is defined to be 52.7 U after extensive calibration. Further, collaborative studies for the evaluation of this serum are needed in order to satisfy the requirements for international reference antiserum.
Vaccine 01/2006; 23(48-49):5666-9. · 3.77 Impact Factor
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Shengli Bi,
E'de Qin,
Zuyuan Xu,
Wei Li,
Jing Wang,
Yongwu Hu,
Yong Liu,
Shumin Duan,
Jianfei Hu,
Yujun Han, [......],
Ruifu Yang,
Bin Liu,
Siqi Liu,
Songgang Li,
Jun Wang,
Jian Wang,
Wuchun Cao,
Jun Yu, Xiaoping Dong,
Huanming Yang
[show abstract]
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ABSTRACT: Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
Genomics Proteomics & Bioinformatics 09/2003; 1(3):180-92.
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ABSTRACT: To establish a method that can quantitatively detect S100 protein in CSF, and evaluate the possibility in diagnosis of Creutzfeldt-Jakob disease (CJD).
S100 gene was amplified by PCR from a commercially supplied human brain cDNA library. After verified by sequence analysis, the full length of S100 DNA was subcloned into a (GST) expression vector Pgex-2T, and the expression of GST-S100 fusion protein was induced. Rabbits were immunized with the purified GST-S100 fusion protein, and the antiserum raised against S100 protein was collected and further evaluated. Using biotin-avidin system, a sandwich ELISA was established for quantitatively determining S100 protein, and further, used in screening for S100 protein in CSF and serum samples.
SDS-PAGE assays yielded a roughly 35,000 GST-S100 fusion protein. Using the established method, three CSF samples from probable CJD patients (14-3-3 protein positive in CSF) showed higher concentration of S100 protein (higher than 2.900 microg/L), whereas other CSF samples collected from patients with other CNS diseases showed lower concentration of S100 (less than 0.180 microg/L).Moreover, the sera S100 proteins from all the collected samples showed distinct individual difference.
The established method can be used in determining S100 protein in CSF quantitatively. The feasibility and significance of S100 protein in CSF for diagnosis of CJD should be further considered with more CSF samples.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 01/2003; 16(4):305-8.
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ABSTRACT: An amino acid polymorphism for Met to Val has been identified at PrP codon 129 from different human races. In this study,the characteristics of polymorphism of PRNP 129th amino acid in Han and Uighur Chinese have been investigated.
Human DNAs were extracted from peripheral lymphocytes and PrP gene fragments were amplified with a specific PCR protocol. The distribution of 129th amino acid in PRNP was determined by a PCR-RFLP and the results were analyzed with software SAS for Windows 6.12.
The frequencies of the allele 129 Met and 129 Val were 97.0% and 3.0% in Han Chinese, whereas 91.4% and 8.6% in Uighur Chinese. The frequency of 129 M/M phenotypes in Han Chinese was significantly higher than that in Uighur Chinese (P=0.0490). Comparing the phenotype distributions of codon 129 of Han Chinese with that of Japanese and Caucasian, there was significant difference with Caucasian (P=0.0005),but there was no difference with Japanese (P=0.5040).
The polymorphism of 129th amino acid in PRNP of Han Chinese is similar to Japanese, but different from Uighur Chinese.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 07/2002; 16(2):105-8.
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ABSTRACT: To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.
The full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T. The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits. The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established. Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro. The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay.
SDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000. The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells. Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method.
The established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities. Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 04/2002; 16(1):69-73.
