Short communicationExpression of the fusion glycoprotein of newcasstle disease virus in transgenic rice and its immunogenicity in mice
Introduction
Newcastle disease virus (NDV), a member of the family Paramyxoviridae, is one of the grievous pathogen of all species of birds and can cause a serious respiratory and neurological disease [1]. In poultry production, the Newcastle disease (ND) can cause a high level of mortality (up to 100%), and has a great negative effect to the surviving birds, such as impaired growth, poor food utilization, reduced egg production, impairment of eggshell formation, and decreased fertility and hatchability of eggs [2]. Thus, ND was considered as one of the hastiest and highest destructive diseases for poultry, especially for chicken, resulted the worldwide trade barriers and dramatically economic losses in the commercial poultry industry.
Vaccination is the primary effective strategy in the prevention of ND in the poultry industry, but the traditional vaccine may cause various problems such as side effects, high labor cost, and stress that may lead to a reduction in egg-laying, or to an increasing susceptibility to microorganisms infections. One of solutions to these problems is to develop the edible vaccines for easy delivery with water or diet [3]. The NDV envelope consists of two important and functional glycoproteins, the fusion (F) protein and hemagglutinin-neuraminidase (HN) protein [4]. The F protein of NDV mediates the fusion of both virus to cell and cell to cell [5], and serves as the key factor for both virulence and immunity. Therefore, it was very attractive to develop the specific ND subunit vaccine by using the NDV F protein as the target, in particular by production of the recombinant NDV F protein in an efficient and economic system [6], [7], [8], [9].
The subunit vaccines are now commercially produced in genetically modified bacteria, yeast or mammalian cells. In the past decades, transgenic plants as bioreactor have been recognized as an effective, safety and economic way to produce recombinant proteins [10]. There is some advantage for the production of recombinant vaccines in transgenic plants, when compared with the ways of traditional vaccine production. For examples, plant is the potential source of antigen protein as it is not dependent upon process technology to ensure protein folding and particle assembly; besides, the plant-based antigen protein production makes it feasible for oral immunization by direct feeding the plant-derived diets. Up to now, several types of viral and bacterial subunit antigen had been successively generated in transgenic plants, including the E. coli heatlabile enterotoxin B subunit (LT-B) [11], [12], [13], [14], [15], [16], [17], cholera toxin B subunit (CT-B) [18], [19], hepatitis B surface antigen (HBsAg) [20], [21], [22], [23], [24], [25], [26], [27], Norwalk virus capsid protein (NVCP) [28], [29], rabies virus glycoprotein [30], etc. And some of the plant-derived antigens had been functionally tested in human or mice, and the results showed that the specific antibody could be detected in sera of the volunteers who were orally or intraperitoneally immunized with recombinant proteins from plants [31].
In present study, we attempted to use rice, the worldwide economic and staple crop for both human food and animal feed, as a novel bioreactor to express the fusion protein of NDV, and to evaluate the immunogenicity of the expressed product from rice. Our results demonstrated that the NDV F protein could be expressed and accumulated in both the vegetable (leaf) and storage (seed) tissue of transgenic rice plants, and the recombinant rice-derived antigen showed good immunogenicity in mice. To our knowledge, this is the first report of using transgenic rice as factory to produce NDV F protein for chicken ND subunit vaccine.
Section snippets
Construction of plant expression vectors
A 1700-bp full-length cDNA encoding the NDV F protein was amplified from a recombinant plasmid pVAX1-NDV-F (Pan et al., unpublished) by PCR with the primer set NDVF-5 (5′-CAGGATCCAATGGGCCCCAAATCTCTAC-3′) and NDVF-3 (5′-CGGAGCTCACATCAT TTTTGTTGTAGTGGCCTCTC-3′), in the 5′ end of both primers, the BamHI and SacI (underlined) site were added, respectively. The PCR product was directly cloned into the vector pGEM-T (Promega) for sequence confirmation, and then digested with both BamHI and SacI to
Generation of transgenic rice plants
The binary vectors pGNDVF and pUNDVF were constructed for the expression of NDV F protein in rice (Fig. 1), and then the expression cassette was introduced into the primary callus derived from mature embryos of rice cultivar GLXN mediated by A. tumefacinens. After three times of selection in the medium containing hygromycin, the resistant calli were obtained in both constructs. The well-growing resistant calli were transferred onto regeneration medium, and after 2–3 weeks the green spot and
Discussion
Comparing with the production systems for traditional recombinant subunit vaccines, which are derived from the genetically engineered bacteria, yeast or mammalian cells, transgenic plant as bioreactor offers a more economic alternative to generate recombinant vaccine antigens [20]. Plant-based antigens are also more safety than those derived from other systems, as there is no chance of contamination of other pathogens. Even more, if the vaccine antigens are expressed in the edible parts of the
Acknowledgements
We thank Mrs. Lu Meifang, Mr. Tang Wei and Dr. Zhang Xiaoming for technical assistance. This work was supported by grants from Fok Ying Tuang Education Foundation (No. 94019), NSFC (No. 30425031, 30470992), and the Government of Jiangsu Province (Nos. BK2003214 and G2002026).
References (47)
- et al.
Newcastle disease vaccine
Biotechnol Adv
(1998) - et al.
The influence of adjuvants on oral vaccination of chickens against Newcastle disease
Vet Microbiol
(1995) - et al.
Role of fusion protein cleavage site in the virulence of Newcastle disease virus
Microb Pathog
(2004) - et al.
Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives
Vaccine
(2003) - et al.
Enternal immunisation and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated inbiodegradable microspheres
Vaccine
(1994) - et al.
Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene
Vaccine
(1998) - et al.
Oral immunization of naive and primed animals with transgenic potato tubers expressing LT-B
Vaccine
(2001) - et al.
Plant-based vaccines: unique advantages
Vaccine
(2001) - et al.
Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts
J Mol Biol
(2001) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)
Plant derived measles virus hemagglutinin protein induces neutralizing antibodies in mice
Vaccine
Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants
Vaccine
Protection of mice against challenge with foot and mouth disease virus (FMDV) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the FMDV structural protein VP1
Virology
Structure and expression of the rice glutelin multigene family
J Biol Chem
Newcastle disease
Identification of biological activities of paramyxovirus glycoproteins
Virology
Virus-cell and cell-cell fusion
Annu Rev Cell Biol
Biopotency of inactivated subunit vaccine of Newcastle disease virus fusion protein produced by gene transfer using a hybrid baculo-virus vector in Bombyxmori mori pupae
Jpn Poult Sci
Newcastle disease virus F glycoprotein expressed from combinant vaccinia virus vector protects chickens against live virus challenge
Avian Pathol
Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence
J Virol
Oral immunization with a recombinant bacterial antigen produced in transgenic plants
Science
Immuno-genicity in humans of a recommbinat bacterial antigen delivered in a transgenic potato
Nat Med
A functional antigen in a practical crop: LT-B producing maize protects mice against Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT)
Transgenic Res
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