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Kim, Chu, Park, Seo, Park, Kim, and Jun: Construction of Recombinant DNA with F and HN Genes of Newcastle Disease Virus and Its Immunogenicity
Original Article

J Bacteriol Virol 2006;36(2):99-107.

Published online: 18 February 2006

DOI: https://doi.org/10.4167/jbv.2006.36.2.99

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

Ji-Young Kim, Jiaqi Chu, Jong-Hyeon Park1, Sang-Heui Seo, Chang-Sik Park2, Myung-Cheol Kim, Moo-Hyung Jun

College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea

1National Veterinary Research and Quarantine Service, Anyang, 430-824, Korea

2Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 305-764, Korea

Received 25 April 2006       Accepted 12 June 2006

Copyright © 2006 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: NDV vaccine, F and HN gene, Recombinant NDV vaccine

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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.
jbv-36-99f1.tif
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.
jbv-36-99f2.tif
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.
jbv-36-99f3.tif
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.
jbv-36-99f4.tif
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.
jbv-36-99f5.tif
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.
jbv-36-99f6.tif
Table 1.
Immunization of the mice against recombinant DNA with F and HN genes
Groups∗ Immunogens No. of mouse Routes Dose at weeks (μg)
0 2 4 6
I pSLIA-tsF 5 IM 100 100 100 Sacrificed
II pSLIA-tsHN 5 IM 100 100 100 Sacrificed
III pSLIA-tsF 5 ID 100 100 100 Sacrificed
IV pSLIA-tsHN 5 ID 100 100 100 Sacrificed
V Control 5 IM 100 100 100 Sacrificed

∗ Mouse: C57BL/6, IM: Intramuscular injection in quadriceps muscle.

ID: Intradermal injection on tail. Control group was injected intramuscularly with pSLIA alone.

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