Expression of Recombinant Fusion Protein from Local Isolate of Newcastle Disease Virus and Antibody Response to Recombinant Fusion Protein in Broiler Chickens Post-Vaccination

This research aimed to express and purify the recombinant Fusion (F) protein of Newcastle Disease Virus (NDV) from a local isolate in Galur, Kulon Progo, Indonesia (0663/04/2013) from recombinant vector plasmid pBT7-N-His F, and to study the antibody response in the broiler sera which were injected with pure recombinant F protein compared with treated broilers that were vaccinated with commercial inactive NDV vaccines and control broilers without vaccination. The results showed that the recombinant F protein of NDV was successfully expressed, purified and visualized by SDS-PAGE with Coomassie Brilliant Blue staining and Westernblotting methods as a specific recombinant F protein with a molecular weight of 28 kDa. The pure recombinant F protein then was injected into broilers to determine the antibody response in broiler serum. Indirect ELISA showed that the production of antibodies was high in F protein vaccinated groups in comparison with other treated and control groups. The recombinant F protein has potential to be developed as a recombinant vaccine candidate after truncating the 6x His-tag part to obtain higher antibody respond if compared with antibody production in broiler serum post vaccinated with some commercially available broiler vaccines.

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Evidence for Mixed Membrane Topology of the Newcastle Disease Virus Fusion Protein

Journal of Virology ◽

10.1128/jvi.77.3.1951-1963.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 1951-1963 ◽

Cited By ~ 27

Author(s):

Lori W. McGinnes ◽

Julie N. Reitter ◽

Kathy Gravel ◽

Trudy G. Morrison

Keyword(s):

Fusion Protein ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Cytoplasmic Domain ◽

Classical Type ◽

F Protein ◽

Amino Terminal ◽

Protease Digestion ◽

A Cell

ABSTRACT The synthesis of the Newcastle disease virus (NDV) fusion (F) protein in a cell-free protein-synthesizing system containing membranes was characterized. The membrane-associated products were in at least two different topological forms with respect to the membranes. The properties of one form were consistent with the expected membrane insertion as a classical type 1 glycoprotein. This form of the protein was fully glycosylated, and sequences amino terminal to the transmembrane domain were protected from protease digestion by the membranes. The second form of membrane-associated F protein was partially glycosylated and partially protected from protease digestion by the membranes. Protease digestion resulted in a 23-kDa protease-protected polypeptide derived from F2 sequences and sequences from the amino-terminal end of the F1 domain. Furthermore, a 10-kDa polypeptide derived from the cytoplasmic domain (CT) was also protected from protease digestion by the membranes. Protease resistance of the 23- and 10-kDa polypeptides suggested that this second form of F protein inserted in membranes in a polytopic conformation with both the amino-terminal end and the carboxyl-terminal end translocated across membranes. To determine if this second form of the fusion protein could be found in cells expressing the F protein, two different approaches were taken. A polypeptide with the size of the partially translocated F protein was detected by Western analysis of proteins in total-cell extracts of NDV strain B1 (avirulent)-infected Cos-7 cells. Using antibodies raised against a peptide with sequences from the cytoplasmic domain, CT sequences were detected on surfaces of F protein-expressing Cos-7 cells by immunofluorescence and by flow cytometry. This antibody also inhibited the fusion of red blood cells to cells expressing F and HN proteins. These results suggest that NDV F protein made both in a cell-free system and in Cos-7 cells may exist in two topological forms with respect to membranes and that the second form of the protein may be involved in cell-cell fusion.

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Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus

Journal of General Virology ◽

10.1099/0022-1317-83-3-623 ◽

2002 ◽

Vol 83 (3) ◽

pp. 623-629 ◽

Cited By ~ 28

Author(s):

Ming Yu ◽

Enxiu Wang ◽

Youfang Liu ◽

Dianjun Cao ◽

Ningyi Jin ◽

...

Keyword(s):

Fusion Protein ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Large Scale ◽

Heptad Repeat ◽

Scale Production ◽

F Protein ◽

Helix Bundle ◽

Fusion Inhibitors

Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein. In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E. coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins. The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex. Furthermore, both the GST fusion protein and the cleaved HR2 show virus–cell fusion inhibition activity (IC50 of 1·07–2·93 μM). The solubility of the GST–HR2 fusion protein is much higher than that of the corresponding peptide. Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors.

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Recombinant fusion protein expression of Indonesian isolate Newcastle disease virus in Escherichia coli BL21(DE3)

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220629 ◽

2021 ◽

Vol 22 (6) ◽

Author(s):

Martha Purnami Wulanjati ◽

Lucia Dhiantika Witasari ◽

Nastiti Wijayanti ◽

Aris Haryanto

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Protein Expression ◽

Newcastle Disease Virus ◽

B Cell ◽

Disease Virus ◽

Newcastle Disease ◽

F Protein ◽

E Coli ◽

Fusion Protein Expression

Abstract. Wulanjati MP, Witasari LD, Wijayanti N, Haryanto A. 2021. Recombinant fusion protein expression of Indonesian isolate Newcastle disease virus in Escherichia coli BL21(DE3). Biodiversitas 22: 3249-3255. Newcastle disease is a major problem in poultry industry due to high mortality of susceptible chicken. New vaccine agents are important to be developed to eliminate the disease threat. This study aimed to examine the expression of fusion (F) protein of Newcastle Disease Virus (NDV) from Indonesian isolates in E. coli BL21(DE3) by IPTG induction. The sample was a part of F gene of NDV from Galur, Kulon Progo, Yogyakarta, Indonesia (0663/04/2013) with a molecular size of 600 bp that was synthesized and inserted into pBT7-N-His expression vector. The recombinant F protein with molecular weight of 25.6 kDa was successfully expressed in E. coli BL21(DE3), purified using Ni-NTA magnetic silica beads, and confirmed by western blotting. Optimization of expression showed that recombinant F protein was optimally expressed by induction of 1.0 mM IPTG when the cells reached OD600 = 0.6. The induction duration was 8 h. B-cell epitopes prediction showed that F protein possessed four epitopes that possibly recognized by B-cell. Since recombinant F protein was considered to possess immunogenicity, its potency as a candidate of NDV vaccine agent should be investigated in the future.

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