title: Construction of an artificial recombinant bicistronic plasmid DNA vaccine against porcine rotavirus.
abstract: The attenuated Salmonella typhimurium χ4550 strain was used to harbour a reconstructed bicistronic DNA vaccine against porcine rotavirus, which carried the rotavirus nonstructural protein 4 (NSP4) and VP7 genes simultaneously. Using a balanced lethal system, the kanamycin resistance gene of expressing eukaryotic plasmids pVAX1 and pVAXD were replaced by the aspartate β-semialdehyde dehydrogenase (asd) gene. The NSP4 cleavage product (259-525) of rotavirus OSU strain and VP7 full-length genes were amplified by reverse transcription polymerase chain reaction and then inserted into the eukaryotic single-expression plasmid, pVAX1-asd, and the eukaryotic dual-expression plasmid, pVAXD-asd, respectively. The recombinant plasmids pVAX1-asd-NSP4, pVAX1-asd-VP7 and pVAXD-asd-NSP4-VP7 were transformed into the attenuated S. typhimurium χ4550 strain by electrotransformation. An indirect immunofluorescence assay of the expressed COS-7 cell suggested that the recombinant S. typhimurium χ4550 strain was constructed successfully. The recombinant S. typhimurium χ4550 strain was orally administered to BALB/c mice. The group immunised with dual- expression plasmids produced a significantly higher level of serum Immunoglobulin G (IgG) and intestinal Immunoglobulin A (IgA) than the group immunised with single-expression plasmids. These results indicated that eukaryotic bicistronic plasmid DNA vaccines could be successfully constructed to enhance humoural, mucosal and cellular immune response against rotavirus infection.
authors.affiliation: College of Veterinary Medicine and Institute of Preventive Veterinary Medicine, Sichuan Agricultural University. csanjie@gmail.com.
authors.forename: Tingting
authors.initials: T
authors.lastname: Cui
authors.forename: Jun
authors.initials: J
authors.lastname: Xiong
authors.forename: Yongzhi
authors.initials: Y
authors.lastname: Wang
authors.forename: Xintian
authors.initials: X
authors.lastname: Wen
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Huang
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Ma
authors.forename: Zhongkai
authors.initials: Z
authors.lastname: Hu
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhao
authors.forename: Sanjie
authors.initials: S
authors.lastname: Cao
mesh_terms: D000818:Animals; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D012401:Rotavirus / Q000276:immunology; D012400:Rotavirus Infections / Q000517:prevention & control / Q000662:veterinary*; D012486:Salmonella typhimurium / Q000235:genetics*; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology*; D014765:Viral Vaccines / Q000008:administration & dosage / Q000276:immunology*
chemical_list: D019444:Vaccines, DNA; D014764:Viral Proteins; D014765:Viral Vaccines