☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

[Construction and the immunogenicity of the recombinant Modified Vaccinia Virus Ankala co-expressing ORF4, ORF5 and ORF6 genes of porcine reproductive and Respiratory Syndrome Virus NJ-a strain].

To develop investigate the recombinant MVA(rMVA) vaccines against PRRSV infection, the ORF4, ORF5 and ORF6 of PRRSV NJ-a strain were subcloned into the MVA transfer vector p II LR and the resultant recombinant vector was called p II LR-ORF4/ORF5/ORF6. The rMVA was generated by transfecting MVA-infected BHK-21 cells with the recombinant vector and screened by plaque purification after X-gal staining. After six rounds of purification, insertion of PRRSV GP4, GP5 and M genes into the MVA genome was confirmed by PCR analysis and expression of the three proteins was identified by Western-blot and IFA. Each of the tested mice was inoculated with 5 x 10(5) TCID(50)/mouse of the rMVA-GP4/GP5/M and boosted 3 weeks later. Neutralization assay showed that PRRSV-specific neutralizing antibodies were detectable at 3 weeks and reached the highest titer (2(5)) by 8 weeks after the primary vaccination, which maintained stable until the end of the experiment. The significant lymphocyte proliferation responses were also observed in mice immunized with rMVA-GP4/GP5/M. These results indicate the rMVA co-expressing PRRSV ORF4, ORF5 andORF6 genes may be an attractive candidate vaccine for preventing PRRSV infection.

[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.