J Bacteriol Virol.  2006 Jun;36(2):99-107. 10.4167/jbv.2006.36.2.99.

Construction of Recombinant DNA with F and HN Genes of Newcastle Disease Virus and Its Immunogenicity

• Kim JY
• Chu J
• Park JH
• Seo SH
• Park CS
• Kim MC
• Jun MH

• KMID: 2055016
• DOI: http://doi.org/10.4167/jbv.2006.36.2.99

Abstract

Recombinant DNA vaccines, based on plasmid vectors expressing an antigen under the control of a strong promotor, have several advantages over traditional vaccines. They have been shown to induce a full spectrum of immune responses for humoral and cellular systems and to secure the higher safety and the simplicity of administration. Thus, establishment of DNA vaccines against Newcastle disease virus (NDV) in poultry has been widely investigated using various virus strains and vector systems. In this study, the F and HN genes of NDV CBP-1 strains isolated from diseased pheasants and attenuated by serial passages in egg embryos were cloned using pSLIA vector and constructed two recombinants of pSLIA-tsF and pSLIA-tsHN. The recombinant plasmids were transfected into COS-7 cell and the expression of HN and F proteins were verified by immunofluorescence, SDS-PAGE and Western blot. The recombinant plasmids were injected intramuscularly and intradermally into C57B/6 mouse and a significant increment of HN and F antibodies was detected by ELISA. According to the results, it was implicative that the recombinant DNA could be utilized for development of recombinant DNA vaccine for NDV.

Keyword

NDV vaccine; F and HN gene; Recombinant NDV vaccine

MeSH Terms

Animals
Antibodies
Blotting, Western
Clone Cells
COS Cells
DNA, Recombinant*
Electrophoresis, Polyacrylamide Gel
Embryonic Structures
Enzyme-Linked Immunosorbent Assay
Fluorescent Antibody Technique
Mice
Newcastle disease virus*
Newcastle Disease*
Ovum
Plasmids
Poultry
Serial Passage
Vaccines
Vaccines, DNA
Antibodies
DNA, Recombinant
Vaccines
Vaccines, DNA

Figure

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  Figure 1. Amplification patterns of NDV F gene (A) and HN gene (B) by the RT-PCR. Lane M: 1 kb DNA ladder marker, lane 1: CBP-1, lane 2: Hitchner B1, lane 3: LaSota-IB strain.

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  Figure 2. Cleavage patterns of F gene (A) and HN gene (B) inserted into pSLIA by various restriction endonucleases. Lane M: 1 kb DNA ladder marker, lane 1: BamHI, lane 2: EcoRI, lane 3: PstI, lane 4: EcoRV.

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  Figure 3. Detection of F and HN proteins expressed by recombinant pSLIA-tsF and pSLIA-tsHN by immunofluorescent assay using monoclonal antibodies. A: Control cells, B: pSLIA-tsF-transfected COS-7 cells, C: pSLIA-tsHN-transfected COS-7 cells.

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  Figure 4. SDS-PAGE patterns of F and HN proteins in COS-7 cell transfected with pSLIA-tsF and pSLIA-tsHN plasmids. Lane M: pre-stained protein marker, lane 1: pSLIA-tsF, lane 2: pSLIA, lane 3: Control, lane 4: pSLIA-tsHN, lane 5: pSLIA, lane 6: Control.

• 

  Figure 5. Western blot analysis of F and HN proteins in COS-7 cell transfected with pSLIA-tsF and pSLIA-tsHN plasmids. Lane M: pre-stained protein marker, lane 1: pSLIA-tsF, lane 2: pSLIA, lane 3: Control, lane 4: pSLIA-tsHN, lane 5: pSLIA, lane 6: Control.

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  Figure 6. Comparison of ELISA values in the mice injected by intramuscular (IM) and intradermal (ID) routes with the pSLIA-tsF and pSLIA-tsHN. ELISA values were measured at 6 weeks after immunization with the plasmids as shown in Table 1. I and II: injected intramuscularly, III and IV: injected intradermally, V (control): injected intramuscularly with pSLIA alone.

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