title: [Construction and immunogenicity of recombinant adenovirus tandem expressing M and GP5 of porcine reproductive and respiratory syndrome virus].
abstract: The M protein gene of porcine reproductive and respiratory syndrome virus amplified by PCR was tandem linked with its GP5 gene in shuttle vector in correct frame, resulting in shuttle vector pShuttle-CMV-M-GP5. The positive clone was identified by PCR and further confirmed by sequencing. The constructed plasmid was linearized with Pme I and co-transformed BJ5183 host bacteria with pAdEasy-1 to produce recombinant adenovirus DNA by homologous recombination. Then the adenovirus DNA was linearized with Pac I and transfected into HEK-293A cells to obtain recombinant adenovirus. The specific expression of target proteins by the recombinant adenovirus was verified by indirect immuno-fluorescence assay (IFA) with monoclonal antibodies against M and GP5.The results showed that the tandem linked M with GP5 could be co-expressed by adenovirus vector. Mice immunized with the constructed recombinant adenovirus induced strong humoral immunity (ELISA antibody and virus neutralizing antibody) and cellular immunity (lymphocyte proliferation and CTL responses). The results showed that the recombinant adenovirus has strong immunogenicity and provided the basis for the further experiments in pigs.
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. jiangp@njau.edu.cn
authors.forename: Wen-Ming
authors.initials: WM
authors.lastname: Jiang
authors.forename: Ping
authors.initials: P
authors.lastname: Jiang
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Li
authors.forename: Jing-Yuan
authors.initials: JY
authors.lastname: Tang
authors.forename: Xian-Wei
authors.initials: XW
authors.lastname: Wang
authors.forename: Yi-Jun
authors.initials: YJ
authors.lastname: Du
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D019316:Porcine respiratory and reproductive syndrome virus / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000276:immunology; D014765:Viral Vaccines / Q000276:immunology
chemical_list: C525999:M protein, porcine reproductive and respiratory syndrome virus; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D014765:Viral Vaccines; C114789:glycoprotein 5, PRRSV