[Influence of epitope A modification and N-linked glycosylated site mutation of PRRSV NJ-a strain ORF5 gene on the ability to induce neutralizing antibodies and T cell proliferation response].
To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.
Zheng
Qi-Sheng
QS
Key Laboratory of Animal Disease Diagnosis and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.
Li
Peng
P
Bi
Zhi-Xiang
ZX
Niu
Ming-Fu
MF
Cao
Rui-Bing
RB
Zhou
Bin
B
Chen
De-Sheng
DS
Chen
Pu-Yan
PY
D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D016466:CHO Cells; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006031:Glycosylation; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D019318:Porcine Reproductive and Respiratory Syndrome / Q000276:immunology / Q000517:prevention & control; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013552:Swine / Q000821:virology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014765:Viral Vaccines / Q000008:administration & dosage / Q000276:immunology
D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D019444:Vaccines, DNA; D014764:Viral Proteins; D014765:Viral Vaccines