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Articles by Yan-Ming Zhang
Total Records ( 5 ) for Yan-Ming Zhang
  Kang-Kang Guo , Wei-Ming Zhang , Yan-Ming Zhang , Peng-Bo Ning , Jing-Yu Wang and Miao-Tao Zhang
  Classical Swine Fever (CSF) is a hemorrhagic disease of pigs caused by virulent strains of Classical Swine Fever Virus (CSFV). Vaccination is a useful tool for preventing and controlling CSF. In this study the protective antigens E0 and E2 genes of CSFV were cloned and inserted into a fowlpox virus vector to construct a recombinant plasmid. We transfected this plasmid into chicken embryo cells infected with the fowlpox virus FV282 to package and propagate the recombinant fowlpox virus FV282-E0-E2. Specific anti-CSFV antibodies were detected with enzyme-linked immunosorbent assay in FV282-E0-E2 immunized experimental mice and pigs. Moreover, virus challenge experiments were conducted in experimental pigs immunized with FV282-E0-E2. Immune protection was evaluated in experimental pigs infected with virulent CSFV by observing clinical manifestations and post-mortem examination. The results showed that recombinant fowlpox virus FV282-E0-E2 was obtained after ten cycles of blue plaque selection. The special anti-CSFV antibodies were detected in the experimental mice and pigs. The immunized pigs could resist the attack of the virulent CSFV strain. The recombinant fowlpox virus FV282-E0-E2 represents a potential candidate for the development of a genetic engineered vaccine for the future prevention of CSF.
  Qian Zhang , Kang-Kang Guo , Yan-Ming Zhang , Chen Dai , Yan-Fen Jiang and Yang-Xin Peng
  MicroRNAs (miRNAs) play important regulatory roles in mammalian cells and viral replication. Let-7 family of microRNA are important regulators in cell differentiation, human cancer and virus infection. The aim of the present study was to investigated miRNA which involved in Classical Swine Fever Virus (CSFV) infected Swine Umbilical Vein Endothelial Cells (SUVECs). Software predicting analysis was performed to screen out the potential miRNA. Four members of let-7 family, let-7a, let-7c, let-7f and let-7i were screened out as candidates. Luciferase activity assay showed that let-7a, let-7c and let-7i could bind to the 3' untranslated regions (3' UTR) of CSFV. Taqman qRT-PCR analyse showed that the inhibition or overexpression of let-7a and let-7c influenced the replication of CSFV. Mimics of let-7a and let-7c could significantly down-regulate CSFV replication. Further study showed that the expression of let-7a and let-7c were down-regulated in the CSFV infected cells and heterogeneous nuclear ribonucleoprotein A1( hnRNP A1), a negative regulator of let-7a, was up-regulated. The results indicated let-7a and let-7c could inhibit viral replication and the CSFV induced negative feedback regulation to interfere with let-7a expression. This study firstly suggested that miRNAs let-7a and let-7c play important roles in the replication of CSFV. These findings provide novel information on the interaction of miRNAs and the replication of CSFV.
  Jian-Ling Liu , Kang-Kang Guo , Zheng-Yuan Su , Xin-Gang Xu , He-Lin Li , Peng-Bo Ning , Hai-Xia Hong , Xiao-Yun Yang and Yan-Ming Zhang
  The viral envelope glycoprotein E2 is major target for eliciting protective antibodies against CSFV in infected animals. To express CSFV E2 protein on eukaryotic cells, the recombinant retroviral vector pBabe-puro-E2 was constructed by inserting full-length CSFV Shimen strain E2 region into pBabe-puro. Both of the recombinant retroviral vector and pVSVg plasmid were transfected eukaryotic 293GP cells using method of calcium phosphate coprecipitation in where thus the recombinant pseudovirus were packaged and propagated. The PK-15 cells were infected with recombinant pseudovirus. The recombinant PK-15 cells were screened by spytomycin resistance and the expression of E2 protein was detected by Flow Cytometer (FCM). The activity of recombinant E2 protein to induce immune responses was evaluated in Balb/c mice and unvaccinated pigs. The results showed that CSFV E2 protein was successfully expressed on PK-15 cells membrane. Anti-E2 antibody induced in experimental animals was detected by Enzyme Linked Immunosorbent Assay (ELISA). Moreover, the virus challenge indicated that the immunized pigs generated effective protection against virulent CSFV. These results indicated that a retroviral-based E2-vaccine is able to induce high level of specific antibodies and exhibits similar protective capability with that induced by the C-strain.
  Kang-Kang Guo , Qing-Hai Tang , Peng-Bo Ning , He-Lin Li , Wei Liu , Qi-Zhuang Lv , Wu-Long Liang , Zhi Lin , Cheng-Cheng Zhang and Yan-Ming Zhang
  The Nonstructural 2 (NS2) protein plays an important role in the infection process of Classical Swine Fever Virus (CSFV), it is an autoprotease cleaving the NS2-3 polyprotein for the NS3 protein release. The NS2 protein is an easily biodegradable protein in cells and the biogradation pathways are not still very clear at present. The Swine Umbilical Vein Endothelial Cell (SUVEC) constucted by the lab is a good cell line model for studying the pathogenic mechanism of CSFV. In this study, the degradation of CSFV NS2 were observed by detecting the NS2 protein expression in SUVEC and PK-15 cells. The recombinant plasmid pEGFP-NS2 with complete NS2 gene of CSFV was transfected into SUVEC and PK-15 cells, respectively. The NS2 RNA was determined by RT-PCR and expression of NS2 protein in cells were analyzed by fluorescence microscopy and western blot assay after transfected. The degradation of NS2 protein also were observed by fluorescence microscopy and Western-blot assay. The results shown that the NS2 protein is short-lived in cells, the biodegradation process could be inhibited by a proteasomal inhibitor (MG132), hinting the NS2 protein is degraded via proteasome pathway in cells and this degradation process was related to the phosphorylation of NS2 protein. This is a primary study on the degradation of CSFV NS2 protein. The future experiments will address the degradation mechanism of CSFV NS2 and find the phosphorylation amino acids of NS2 protein which maybe relate to protein degadation in cells.
  Bao-Yu Chen , Kang-Kang Guo , Jin-Jin Wang , Lei He and Yan-Ming Zhang
  The tracheal epithelium is an important barrier that protects against harmful inhaled substances. To facilitate understanding of the mechanisms underlying respiratory diseases including tracheal cancer and tracheitis, researchers established an immortalized tracheal epithelial cell line. Primary cultures of Swine Tracheal Epithelial Cells (STECs) were immortalized by transfection of human Telomerase Reverse Transcriptase (hTERT; pCI-neo-hTERT) using lipofection. Positive cells were selected with G418 and expanded for continuous culture for up to 60 passages. The expression of hTERT mRNA in transfected cells was detected by RT-PCR. Transfected cells were assessed for morphology, karyotype, growth in soft agar and tumorigenicity in nude mice. Immortalized cells showed similar properties to those of normal cells such as contact inhibition, serum requirement and anchorage dependence. A soft agar assay and karyotype analysis showed no neoplastic transformation. These results suggest that immortalized STECs induced by the hTERT gene retain their original characteristics. The immortal STEC line may be useful as an in vitro model of tracheal epithelium for physiological, pathological and pharmacological investigations.
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