Abstract
The haemagglutinin-neuraminidase (HN) gene ofNewcastle disease virus (NDV) strain AF2240, amplifiedfrom the viral genomic RNA (∼1.8 kb) was directionallycloned and inserted into a baculovirus expressionvector system. The recombinant glycoprotein expressedin Spodoptera frugiperda (Sf9) cellsshowed haemagglutinin (HA), neuraminidase (NA) andhemadsorption activities. HA activity was detected inboth extra- and intra-cellular recombinant HN(recHNAF2240) samples. In addition, both HA andhemadsorption activities were inhibited by polyclonalanti-NDV sera. Furthermore, significant expression ofthe recombinant protein was observed on the surface ofinfected cells. SDS-PAGE analysis revealed thepresence of visually distinguishable bands between the70 and 80 kDa in size that were absent in thewild-type samples. Western blot analysis showed thatthe distinct ∼63 kDa band and a ∼75 kDa bandcorresponded to the unglycosylated and glycosylated HNglycoprotein respectively as reported in anotherstudy. These observations indicated that the HNrecombinant protein was not only expressed on thesurface of the infected cells as well as with theviral coat protein, but also appears to be functional.
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References
Aminoff D (1961) Methods for the quantitative estimation of N-Acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J 81: 384-392.
Chulan U, Ibrahim AL, Babjee AM & Sheikh-Omar AR (1982) Vaccination against Newcastle disease. Trop Anim Health Prod 14: 177-184.
Coelingh KLV, Murphy BR, Collins PL, Lebacq-Verheyden AM & Battey JF (1987) Expression of biologically active and antigenically authentic parainfluenza type 3 virus hemagglutininneuraminidase glycoproteins by a recombinant baculovirus. Virology 160: 465-472.
Ernawati R & Ibrahim AL (1984) Newcastle disease vaccination in Malaysia. Application of oil emulsion vaccine. Vet Rec 115: 330-339.
Ghiasi H, Kaiwar R, Nesburn AB & Wechsler SL (1992) Expression of herpes simplex virus type I glycoprotein in baculovirus: Preliminary biochemical characterization and protection studies. J Virol 66: 2505-2509.
Ghiasi H, Nesburn AB & Wechsler SL (1991) Cell surface expression of herpes simplex virus type 1 glycoprotein H in recombinant baculovirus-infected cells. Virology 185: 187-194.
Ghiasi H, Slanina S, Nesburn AB & Wechsler SL (1994) Characterization of baculovirus-expressed herpes simplex virus type 1 glycoprotein K. J Virol 68: 2347-2354.
Ibrahim AL, Chulan U & Babjee AM (1980) The immune response of chickens against Newcastle disease with Newcastle disease V4 vaccine. Aust Vet J 56: 29-33.
Ideris A, Ibrahim AL & Spradbrow PB (1990) Vaccination of chickens against Newcastle Disease Virus with a Food Pellet Vaccine. Avian Pathol 19: 371-384.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685.
Mackow ER, Barnett JW, Chan H & Greenberg HB (1989) The rhesus rotavirus outer capsid protein VP4 functions as a hemagglutinin and is antigenically conserved when expressed by a baculovirus recombinant. J Virol 63: 1661-1668.
Marz L, Altmann F, Staudacher E & Kubelka V (1995) Protein glycosylation in insects. In: Montreuil J, Vliegenthart JFG & Schachter H (eds) Glycoproteins (pp. 543-563) Elsevier Science B.V., The Netherlands.
Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA & Summers MD (1995) Virus Taxonomy. Classification and Nomenculture of Viruses. Sixth Report of the International Committee on Taxonomy of Viruses. Archives of Virology [Supplement] 10 (pp. 268-274) Springer, Vienna, New York.
Nagy E, Derbyshire JB, Dobos P & Krell PJ (1990) Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system. Virology 176: 426-438.
Nagy E, Huber P, Krell PJ & Derbyshire JB (1991) Synthesis of Newcastle disease virus (NDV)-like envelopes in insect cells infected with a recombinant baculovirus expressing the hemagglutinin-neuraminidase of NDV. J Gen Virol 72: 753-756.
Nishikawa K, Morishima T, Toyoda T, Miyadai T, Yokochi T, Yoshida, T & Nagai Y (1986) Topological and operational delineation of antigenic sites on the HN glycoprotein of Newcastle disease virus and their structural requirements. J Virol 60: 987-993.
Ong HKA, Ali AM, Omar AR, Tan WS & Yusoff K (1999) N-linked Glycosylated HN Protein of NDV Strain AF2240 expressed in Baculovirus-infected SF9 cells. J Biochem Mol Biol Biophys 3: 147-151.
O'Reilly DR, Miller LK & Luckow VA (1992) Baculovirus Expression Vectors: A Laboratory Manual. WH Freeman and Company, New York, Ottawa Press, Canada.
Rossier E, Miller HR & Phipps PH (1989) Rapid Viral Diagnosis by Immunofluorescence: An Atlas and Practical Guide. University of Ottawa Press, Canada.
Sanger F, Nicklen S & Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467.
Sakaguchi T, Toyoda T, Gotoh B, Inocencio NM, Kuma K, Miyata T & Nagai Y (1989) Newcastle disease virus evolution: I. Multiple lineages by sequence variability of the Hemagglutininneuraminidase gene. Virology 169: 260-272.
Schaper UM, Fuller FJ, Ward MDW, Mehrotra Y, Stone HO, Stripp BR & Debuysscher EV (1988) Nucleotide sequence of the envelope protein genes of highly virulent, neurotropic strain of Newcastle disease virus. Virology 165: 291-295.
Scheid A & Choppin PW (1973) Isolation and purification of envelope protein of NDV. J Virol 11: 263-271.
Tan WS, Lau CH, Ng BK, Ibrahim AL & Yusoff K (1995) Nucleotide sequence of the hemagglutinin-neuraminidase (HN) gene of Malaysian heat resistanct viscerotropic-velogenic Newcastle disease virus (NDV) strain AF2240. DNA Seq 6: 47-50.
Vidal S, Mottet G, Kolakofsky D & Roux L (1989) Addition of high mannose sugars must precede disulfide bond formation for proper folding of Sendai virus glycoproteins. J Virol 63: 892-900.
Wisdom GB (1994) Protein blotting. In: John MW (ed) Methods in Molecular Biology: Basic Protein and Peptide Protocols, Vol. 32 (pp. 207-213). Humana Press, Totowa, New Jersey.
Yusoff K, Tan WS, Lau CH, Ng BK & Ibrahim AL (1996) Sequence of the hemagglutinin-neuraminidase gene of Newcastle disease virus oral vaccine strain V4(UPM). Avian Pathol 25: 837-844.
Yusoff K, Nesbit M, McCartney H, Emmerson PT & Samson ACR (1988) Mapping of three antigenic sites on the hemagglutininneuraminidase protein of Newcastle disease virus. Virus Res 11: 319-333.
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Ong, H.K.A., Ali, A.M., Omar, A.R. et al. Cloning and expression of the HN gene from the velogenic viscerotropic Newcastle disease virus strain AF2240 in Sf9 insect cells. Cytotechnology 32, 243–251 (2000). https://doi.org/10.1023/A:1008136326756
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DOI: https://doi.org/10.1023/A:1008136326756