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Articles
by
He-Lin Li |
Total Records (
2 ) for
He-Lin Li |
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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
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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. |
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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
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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. |
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