Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology

The Role of the N-linked Glycosylation in the prM-E Protein of JEV

Author ZaiJunJie
Tutor ChenHuanChun
School Huazhong Agricultural University
Course Preventive Veterinary Medicine
Keywords Japanese encephalitis virus N- glycosylation prM-E protein Fold Secretion Assembly
CLC S852.65
Type Master's thesis
Year 2011
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Japanese encephalitis virus is an important zoonotic infectious pathogen. The virus is not only against the human nervous system, resulting in the deaths of adults and children, but can also be caused by porcine reproductive disorders, causing huge economic losses to the pig industry each year. The virus is mainly popular in the vicinity of South Asia, Southeast Asia and East Asia, in recent years, expanding in Australia and Russia has some of the reported cases of Japanese encephalitis-prone area. Japanese encephalitis prevention and control rely mainly on current vaccinations, lack of effective therapies. N-glycosylation in protein processing and transport processes play an important role in recent years, a growing number of studies have shown that N-glycosylation on the virulence of the virus replication, infection and immunity and so has important biological school functions. prM and E are two important structural protein of the Japanese encephalitis, both forming the prM-E heterodimers, and can be assembled into virus-like particles in the receptor binding, membrane fusion, neurovirulence, perineural invasion force plays a major role, at the same time can induce the production of neutralizing antibodies. The Japanese encephalitis virus prM and E proteins contain an N-glycosylation sites in the 15 and 154 amino acid residues at the N-glycosylation sites of prM protein in the flavivirus genus are highly conserved, whereas the E protein of the N-glycosylation sites has a certain variation. -N-sugar for the Japanese encephalitis virus study also did not carry out. In this thesis, the N-glycosylation sites in prM and E proteins of Japanese encephalitis virus E protein folding, secretion, and the formation of virus-like particles and cells virulence. Mainly obtained following the results: 1.prME glycosylation mutation expression plasmid amplification of the Japanese encephalitis virus prME genes connected to the gene eukaryotic expression plasmid pcDNA3.1 () construct the plasmid pcprME. The use of PCR-mediated site-directed mutagenesis, respectively, of asparagine in the prM and E proteins are mutated to glutamic acid, N-glycosylation sites eliminating gene. Will be connect mutant prME genes into the eukaryotic expression vector pcDNA3.1 () construct the plasmid mcprME15, mcprME154 mcprME15154, sequencing results showed that all point mutations success. The plasmid were transfected HEK-293FT cells, efficient expression of the prM and E proteins was confirmed by indirect immunofluorescence assay. The expressed protein, Western Blot analysis, the results show the lack of N-glycosylation sites of the prM and E proteins in SDS-PAGE migration rate is greater than the wild-type protein, indicating that point mutations in the prM and E proteins N-sugar glycosylation sites were successfully eliminated. Collapsed 2.N-glycosylation of E protein eukaryotic expression plasmid pcprME, mcprME15 mcprME154 and mcprME15154 were transfected HEK-293FT cells as an anti-space E protein antibody, indirect immunofluorescence test showed that when the prM protein N-Glycosyl groups of bits point deletion, plasmid mcprME15 and mcprME15154 transfected cells, all expression of the E protein are not obtained the correct spatial configuration as described prM protein N-sugar group of the E protein of The correctly folded decisive role. When the deletion of the N-glycosylation sites of the protein E, plasmid mcprME154 expression of most of the E protein is not properly folded, only a small amount of the E protein to obtain the correct spatial conformation. Has an important role of the E protein of the N-glycosylation for its folding, although can also occur in the absence of the protein properly folded, but the folding efficiency is greatly reduced. 3.N-glycosylation of the E protein secretion the plasmid pcprME, mcprME15, mcprME154 and mcprME15154 transfected HEK-293FT cells, collecting the supernatant of the cells. The results showed that the content of the E protein of the plasmid pcprME supernatant far higher than mcprME the 15, the mcprME 154 and mcprME15154 supernatant E protein content with a double antibody sandwich ELISA method to detect the content of the supernatant of the E protein. E protein secretion prM or E protein N-sugar-based sites missing will be greatly affected. 4.N-glycosylation of prM and E protein formed virus-like particles the plasmid pcprME, mcprME15 mcprME 154 and mcprME15154 transfected HEK-293FT cells, fixed cells with 2.5% glutaraldehyde. The number of cells were observed under the electron microscope slice, and finally within the transfected cells the plasmid pcprME mcprME 154 found that the virus-like particles, while in the mcprME, mcprME15154 transfected cell even if the increase in cell slice is not to the virus-like particles were observed. Of N-glycosylation in the prM protein has a decisive influence on the formation of virus-like particles, while the N-glycosylation sites in the E-protein does not have a decisive role in the formation of virus-like particles, but it generates efficiency has a certain impact. Of 5.prM and E protein N-glycosylation of cytopathic effect plasmid pcprME mcprME15 mcprME 154 and mcprME15154 were transfected HEK-293FT cells, trypsin digestion, cells were collected, using the Annexin V-FITC cell withered death detection kit apoptosis ratio. The results showed that the plasmid pcprME (9.38%) transfected cells caused apoptosis rate to be higher than mcprME15 (2.00%), mcprME154 (3.74%), and mcprME15154 (1.30%). Illustrates the N-glycosylation affects the cytotoxicity of the prM and E proteins.

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