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Articles by Chuangfu Chen
Total Records ( 9 ) for Chuangfu Chen
  Shengwei Hu , Wei Ni , Wureli Hazi , Hui Zhang , Na Zhang , Ren Meng and Chuangfu Chen
  Gene silencing mediated by small interfering RNA has become a powerful biological tool for the regulation of gene expression. In order to develop an effective short hairpin RNA (shRNA) expression vector, specifically for use in sheep species, we have identified two sheep U6 promoters based on the highly conserved polymerase III promoter elements. Promoter activity was measured by U6 promoter-driven shRNA to suppress enhanced green fluorescent protein (EGFP) expression. The knock down assay demonstrated that the two sheep U6 promoters and mouse U6 promoter induced a similar level of EGFP knockdown. These results suggest that the two sheep U6 promoters could efficiently drive shRNA expression for gene silencing and may have applications in RNAi-based sheep research.
  Jinliang Sheng , Chuangfu Chen , Xia Yang , Yuanzhi Wang , Pengyan Wang and Hui Zhang
  In this study, Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) assays were performed with EvaGreen to investigate the dynamics of cytokine (Interleukin (IL)-1β, IL-8, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interferon (IFN) (γ) and Toll-Like Receptor 2/4 (TLR2/4) gene expression in ovine primary Alveolar Macrophages (AMs) following Lipopolysaccharide (LPS) stimulation. Expression of cytokine and TLR2/4 mRNA was quantified by comparison of Cycle threshold (CT) values with a standard curve generated from plasmid DNA containing the target gene. Examination of LPS-stimulated ovine AMs revealed that cytokine mRNA expression peaked between 4 and 12 h with the exception of IFN-γ mRNA which peaked around 16 h post stimulation. Furthermore, TLR2 and TLR4 mRNA expression rapidly increased post-stimulation and peaked 20 min post-stimulation at a level which was maintained throughout the procedure. In summary, a sensitive and reliable real-time RT-PCR protocol was implemented for the analysis of ovine TLR2/4 and cytokine gene expression profiles.
  Wureli Hazi , Shengwei Hu , Wei Ni , Zhirui He , Ren Meng , Chuangfu Chen and Ningying Xu
  Foot and Mouth Disease Virus (FMDV) is one of the most important pathogen to the cattle industry often resulting in severe economic losses. Researchers have reported previously a therapeutic application of plasmid-based shRNA against FMDV but the high degree of sequence diversity between different FMDV serotypes may result in the appearance of escape mutants. In this study, a dual shRNA expression plasmid which can simultaneously express two different shRNA molecules was established and showed stronger inhibitory effects on virus replication than the mixture of two shRNAs. Moreover, the antiviral activity induced by the dual shRNA expression system was aslo evident on other FMDV serotypes. Therefore, the dual shRNA system targeting two conserved regions of virus genome provides a more powerful strategy for inhibiting FMDV replication in a cross-resistance manner and implicates a potential application in the treatment of high genetic variability of FMDV.
  Shengwei Hu , Wei Ni , Chuangfu Chen , Wujiafu Sai , Wureli Hazi , Zhirui He , Ren Meng and Jixing Guo
  The present study was carried out to assess and compare the effects of Valproic Acid (VPA) and Trichostatin A (TSA) on in vitro development of sheep Somatic Cell Nuclear Transfer (SCNT) embryos. The results showed that treatment of cloned sheep embryos with 4 mM VPA or 50 nM TSA for 24 h after activation could significantly improve blastocyst rate compared to the control (30.7 vs. 23.3 vs. 16.7%, respectively p<0.05). VPA treatment resulted in a significant higher blastocyst rate than that of TSA-treated group (p<0.05). Moreover, VPA treatment significantly increased (p<0.05) total cell number per blastocyst compared with the TSA treatment and control groups (78.8±9.3 vs. 69.6±9.7 vs. 64.1±8.6, respectively). Furthermore, VPA treatment increased expression of the development-related genes OCT4 and SOX2 in SCNT blastocysts. These results demonstrate that VPA may be more potent than TSA in supporting developmental competence of cloned embryos.
  Fei Guo , Yuanzhi Wang , Chuangfu Chen , Hui Zhang , Jun Qiao , Yan Ren , Junbo Zhang and Zhiqiang Li
  TFSS is an important virulence factor of Brucella, organized as one operon containing 12 different across the cell wall bacterial proteins among which VirB5 regulates the host phagocytosis of Brucella and the transportation of Brucella in the host cells. This study has constructed cDNA library from Brucella melitensis 16M-infected murine macrophage Raw264.7, identified and confirmed the interaction between Brucella VirB5 and FTH1 of RAW264.7 using yeast two-hybrid and Co-Immunoprecipitation (Co-IP) technologies. Subsequently, the morphological changes and the expression of apoptosis-related genes in Brucella-infected RAW264.7 cells have been investigated with Electron Microscope (EM) and real-time quantitative RT-PCR, respectively. The present study has demonstrated that VirB5 and FTH1 play important roles in intracellular parasitism of Brucella and inhibition of FHT1 expression accelerates the apoptosis of macrophage.
  Wenge Hu , Shiwei Ma , Chuangfu Chen , Yuanzhi Wang , Yan Ren , Xudong Cao and Jinliang Sheng
  Leuciscus merzbacheri is a unique vulnerable indigenous fish which is only distributed in the Junggar Basin, Xinjiang. In this study, researchers cloned two L. merzbacheri β-actin promoter fragments of different length: SZ11 and SZ21 and analyzed their structural features. The mammalian expression vector pEGFP-N1 was used to construct eukaryotic expression vectors β1 pEGFP-N1-AFP III and β2 pEGFP-N1-AFP III in which fish type III antifreeze protein gene was used as the structural gene. The results showed that the cloned two fragments of β-actin promoters had the ability to drive the expression of the green fluorescent protein gene in BHK-21 cells. These data suggest that the vectors researchers constructed based on β-actin promoter could be exploited as all fish recombinant eukaryotic expression vector.
  Yuanzhi Wang , Ke Zhang , Yali Zhang , Hui Wang , Fei Guo , Lin Zhang , Hui Zhang , Lijuan CaoBuyun , Cui , Chengyao Li , Li Yuan , Wanjiang Zhang , Ze Xu and Chuangfu Chen
  An outbreak of brucellosis occurred in students on field practice at sheep farm in 2005 at Shihezi, Xinjiang Province, the North-West of China. Five of 7 (71.4%) students were seropositive, showing titers ≥1:160 IU mL-1 in STAT and diagnosed as acute brucellosis with physical examination. To characterize Brucella isolates from the outbreak, the research including face to face investigation, Brucella isolation, multiple locus VNTR-16 analysis (MLVA-16) and genome sequencing were carried out. The investigation showed 42.5% (1,293/3,042) of ewes are sero-positive with RBPT and almost half of ewes aborted. Although, no bacteria were isolated from student blood samples, five individual colonies were isolated from aborted sheep fetuses and were identified as B. melitensis biovar 3 by conventional microbiological tests. MLVA-16 typing indicated that the isolates were clustered in the East Mediterranean with genotype 42. They were similar to wild strains from Guangdong in 2008 and Inner Mongolia in 1994 and 1995. They were most close to strain bru0261 from Pakistan student studying in Germany. Genome sequence and phylogenomic tree showed that pathogen in this study was close to Chinese wild and vaccine strains such as B. melitensis M28, M5-90. Researchers first report pathogens isolated in 1980 and 2005 are genotype 42 containing novel MLVA-16 patterns (1-5-3-13-2-2-3-2-4-20-8-8-4-3-7-7) compared to that both in China and other countries.
  Fei Guo , Junbo Zhang , Chuangfu Chen , Yuanzhi Wang and Hui Zhang
  Brucellosis is an infectious disease that brings great economic burdens for developing countries. The vaccine S2 which is an attenuated Brucella suis (B. suis) strain has been used on a large scale in China. However, the immunity induced by S2 declined relative to those vaccinated with Rev-1 and S19 vaccines. Moreover, the vaccine S2 cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent B. suis vaccine is needed. In this study, a vjbR mutant of B. suis (B. suisΔvjbR) was constructed overcome these drawbacks. The B. suisΔvjbR strain showed reduced survival capability in RAW264.7 macrophage and mice and induced high protective immunity in mice. In addition, B. suisΔvjbR induced an anti-Brucella-specific IgG (immunoglobulin G) response and stimulated the expression of gamma interferon (INFγ). Further, the vjbR antigen allowed serological differentiation between natural and vaccinated infection in mice. Therefore, B. suisΔvjbR was suggested as a safe and efficacious live vaccine candidate against virulent Brucella suis (B. suis) infection.
  Wolong Ma , Fei Guo , Antao Wang , Zhanhui Song and Chuangfu Chen
  A rifampin-resistant mutant of Brucella abortus, designated as RB51 was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. In this study, 325 Mb data was produced for RB51 using Illumina HiSeq 2000 sequencing platform. Based on the assemble result of RB51, researchers obtained that 36 scaffolds with GC content of 57.25% which contains 150 contigs and has genome size was about 3.26 Mb. Detail bioinformatics annotation of RB51 revealed that the genome contained 3,283 genes, the total length of genes was 2,797,347 bp which makes up 85.91% of genome. Comparative genomics analysis found that there was a tandem repeat sequence of 339 nucleotide in YP_413569.1 protein. In addition, RB51 encoded a glycosyltransferase wboA in scaffold12, an enzyme essential in the biosynthesis of O antigen which has been disrupted by IS711. In comparison with the reference genome B. abortus 2308, researchers found that RB51 contained 135 SNPs and 5 InDels among which 1 SNP and 2 InDels may be associated with virulent related to lipopolysaccharide.
 
 
 
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