Expression of full length fusion (F) protein gene of Newcastle disease virus in mammalian expression system

S MANJUNATH; K VIJAYARANI; K KUMANAN

doi:10.56093/ijans.v84i4.39830

Authors

S MANJUNATH Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 007 India
K VIJAYARANI Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 007 India
K KUMANAN Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 007 India

https://doi.org/10.56093/ijans.v84i4.39830

Keywords:

Fusion gene, Newcastle disease virus, Immunofluorescence assay, Mammalian expression, Prokaryotic expression, SDS PAGE, Sub-cloning pTriEx, TOPO cloning, Western Blot

Abstract

The present study was carried out to clone and express fusion protein gene of Newcastle disease virus in mammalian expression system. The Newcastle disease virus (2K3) isolate from pigeon was propagated in 9–11 day-old embryonated chicken eggs and infected allantoic fluid was collected for RNA isolation. The fusion (F) gene of 1.68 Kb was amplified with cDNA as template and was cloned in TOPO cloning vector. The recombinant TOPO colonies were digested with NcoI and XhoI enzymes, the insert released was further ligated into pTriEx Neo 1.1 expression vector digested with the same enzymes and was transformed in Escherichia coli. The recombinant pTriEx colonies confirmed by colony PCR and restriction digestion were induced with 1mM IPTG which showed expressed fusion protein of 55 kDa at 4 h post induction and increased in overnight induced cultures. The recombinant pTriEx plasmid was transfected into vero cells. The cell lysate collected at 48 h and 72 h post transfection showed expressed fusion protein with the molecular weight of 55 kDa in 12% SDS-PAGE. The protein was further confirmed to be NDV specific by its immunoreactivity with NDV antiserum raised in rabbits showing fusion protein of 55 kDa. The immunofluorescence assay of transfected vero cells exhibited a bright cytoplasmic fluorescence confirming the fusion protein expression.

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References

Aldous E W, Collins M S, Goldrick A M and Alexander D J. 2001. Rapid pathotyping of newcastle disease virus (NDV) with fluorogenic probes in a PCR assay. Vetrinary Microbiology 80: 201–12.

Alexander D J. 2000. Newcastle disease and other avian paramyxoviruses. Revue scientifique et technique (International Office of Epizootics) 19: 443–62.

Berinstein A, Sellers H S, King D J and Seal B S. 2001. Use of heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates. Journal of Clinical Microbiology 39: 3171–78.

Chambers P, Millar N S, Bingham R W and Emmerson P T. 1986. Molecular cloning of complementary DNA of Newcastle disease virus and the nucleotide sequence analysis of the junction between the genes encoding haemagglutinin neuraminidase and the large protein. Journal of General Virology 67: 475–86.

Creelan J L, Graham D. A and McCullough J. 2002. Detection and differentiation of pathogenecity of avian paramyxoviruses serotype-1 from field cases with one-step reverse transcriptase polymerase chain reaction. Avian Pathology 31: 493–99.

Kim S H, Wanasen N, Paldurai A, Xiao S, Collins P L, Samal S K. 2013. Newcastle disease virus fusion protein is the major contributor to protective immunity of genotype-matched vaccine. PLOS one 8: e74022.

Lee Y J, Sung H W, Choi J G, Lee E K, Yoon H, Kim J H and Song C S. 2008. Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing fusion and haemagglutinin neuraminidase proteins. Journal of Veterinary Science 9: 301–08.

Lien Y Y, Lee J W, Pu H Y, Tsai H J, Hsieh C Y and Tsai S S. 2007. Phylogenetic characterization of Newcastle disease viruses isolated from Taiwan during 2003–2006. Veterinary Microbiology 123: 194–202.

Liu D, Wang J and Niu Z X. 2010. Contribution of chinese peckin duck complement component C3d-P29 repeats to enhancemnent of Th-2 biased immune responses aganist NDV- F gene induced by DNA immunization. Journal of Immunopathology and Immunotoxicology 32: 297–306.

Loke C F, Omar A R, Raha A R and Yousuff K. 2005. Improved protection from Newcastle disease virus challenge following multiple immunizations with and HN genes. Veterinary Immunology and Immunopathology 106: 259–267.

Millar N S, Chambers P and Emmerson P T. 1988. Nucleotide sequence of the fusion and haemagglutinin neuraminidase glycoprotein genes of Newcastle disease virus, strain Ulster: molecular basis for variations in pathogenecity between starins Journal of General Virology. 69: 613–20.

Miller P J, Decanini E L and Afonso C L. 2010. Evolution of genotypes and related diagnostic challenges. Infection, Genetics and Evolution 10: 26–35.

Nagai Y. and Klenk H D. 1977. Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage. Virology 77: 125–34.

Patel, C., A. K. Tiwari, R. S. Kataria, P. K. Gupta, J. Kataria and A. Rai. 2008. Immunization of birds with recombinant plasmid DNA containing F gene from virulent Newcastle disease virus gives better protection as compared to F gene from avirulent strains against lethal challenge. Journal of Immunology and Immunopathology 10: 20–24.

Rajawat, Y S, Sundaresan N R, Ravindra P V, Kantaraja C, Ratta B,. Sudhagar M, Rai A, Saxena V K, Pilai S K and Tiwari A K. 2008. Immune responses induced by DNA vaccines encoding Newcastle disease virus HN and F proteins in maternal antibody positive commercial broiler chicken. British Poultry Science 49: 111–17.

Römer- Oberdörfer A, Werner O, Veits J, Mebatsion T and Mettenleiter T C. 2003. Contribution of thelength of the HN protein an the sequence of the F protein cleavage site to Newcastle disease virus pathogenecity. Journal of General Virology 84: 3121–29.

Scheid A. and Choppin P W. 1977. Two disulfide linked polypeptide chains constitute the active F protein of Paramyxoviruses. Virology 80: 54–66.

Wilde A, Mc Quain C and Morrison T. 1986. Identification of the sequence content of four polycistronic transcripts synthesized in Newcastle disease virus infected cells. Virus Research 5: 77–95.

Wise M G, Suarez D L, Seal B S, Pedersen J C, Senne D A, King D J, Kapczynski D R and Spackman E. 2004. Development of real time reverse transcription PCR for the detection of Newcastle disease virus RNA in clinical samples. Journal of Clinical Microbiology 42: 329–38.

Yusoff K and Tan W S. 2001. Newcastle disease virus: macromolecules and opportunities. Avian Pathology 30: 439– 55.