Expression of the fusion glycoprotein of newcasstle disease virus in transgenic rice and its immunogenicity in mice

Abstract

Transgenic plant has become an attractive bioreactor to produce high-value medical peptides and proteins in biomedical research. In present study, two expression cassettes, pUNDVF and pGNDVF containing the fusion protein gene of Newcastle disease virus (NDV F) under the control of maize ubiquitin (Ubi) promoter or rice glutelin (Gt1) promoter, respectively, were constructed, and introduced into rice (Oryzy sativa L.) by Agrobacterium-mediated transformation. A total of 12 independent transgenic rice lines were regenerated, and the result from PCR analysis indicated that the T-DNA region containing the NDV F chimeric gene had been integrated into the genome of transgenic rice plants. ELISA and Western-blot analyses revealed that the NDV F protein could be expressed and accumulated in both leaf and seed tissue of several transgenic rice plants. Moreover, the immunogenicity of expressed proteins was tested in a mouse model and the results showed that specific antibodies were elicited in mice immunized intraperitoneally with crude protein extracts from transgenic rice plants. It implied the potential of using transgenic rice-based expression systems as supplementary bioreactor for NDV engineering subunit vaccine.

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.