Expression of hemagglutinin-neuraminidase protein of Newcastle disease virus in transgenic tobacco

Abstract

Newcastle disease is one of the most important pathogens of domestic poultry including chickens. The hemagglutinin-neuraminidase (HN) of Newcastle disease virus is the principal target of neutralizing and protective antibodies against Newcastle disease. In this paper, we transformed tobacco plants with Agrobacterium tumefaciens EHA105 to generate the plants expressing HN of Newcastle disease virus (NDV). The insertion and copy numbers of HN gene in the genomic DNA of phosphinothricin-resistant plants were confirmed by PCR and Southern blot, respectively. The presence of the HN-specific transcript in the total RNAs of the leaves of transgenic plants was verified by Northern analysis. The recombinant HN proteins were detected by Western blot analysis using a polyclonal antibody against GST-HN fusion proteins. The highest level of expression of HN in leaves of transgenic plants was approximately 0.069% of the total soluble protein. ELISA assay showed that the recombinant protein extracted from transformants has normal immunoactivity. Transgenic tobacco expressing HN of NDV with sterilized PBS was fed to 6-week-old chickens. Immunized chickens developed slightly high titers of anti-HN serum IgG compared with those of the wild type plant. These results suggest that oral immunization with HN-transgenic tobacco provides a potential means of protecting chickens from NDV. Further modified animal experiments would be needed to increase the immunity of HN by co-administration of classical adjuvants or other trials.

Production of vaccines for treatment of infectious diseases by transgenic plants

“…The main candidates for developing a vaccine against the disease are fusion protein (F) and hemagglutiningneuraminidase protein (HN) since they are both exposed on the surface of the virus and take part in the infection. HN protein has been expressed in N. benthamiana Lai et al, 2013), potato (Berinstein et al, 2005) and tobacco (Hahn et al, 2007), F protein has been expressed in transgenic rice , potato (Berinstein et al, 2005) and maize (GuerreroAndale et al, 2006). These experiments proved that HN and F proteins can be successfully expressed in transgenic plants and that obtained proteins are immunogenic when delivered to either mice or chickens.…”

“…These experiments proved that HN and F proteins can be successfully expressed in transgenic plants and that obtained proteins are immunogenic when delivered to either mice or chickens. Both maize-produced NDV F protein (Guerrero-Andrade et al, 2006) and tobacco-produced HN protein (Hahn et al, 2007) have been found to be immunogenic in orally immunized chickens; oral immunization with F protein gave 100 % protection of chickens against nasal challenge with NDV, while in the case of HN protein protection against nasal NDV challenge failed but the authors reported a big increase in anti-HN serum IgG levels 28 days after vaccination, increased by 2-fold in 40 % of examined chickens and 4-fold in 20 % of examined chickens (Hahn et al, 2007). HN and F proteins, co-expressed in potato, have been shown to elicit anti-NDV antibodies in intraperitoneally immunized mice and serum IgG at levels comparable to those elicited in mice fed with nontransformed plants soaked in NDV (Berinstein et al, 2005), while the intestinal fluids of those mice also showed considerable IgA and IgG levels.…”

“…F protein, stably expressed under ubiquitin promoter or rice glutelin promoter in rice, elicited anti-NDV serum IgG in intraperitoneally immunized mice . From the point of view of expression level for each of the expressed protein it was noted that F protein was expressed higher in maize kernels than in rice (0.9 -3 % TSP and 0.0025 -0.0055 % TSP, respectively) (GuerreroAndrade et al, 2006;Yang et al, 2007), while the expression of HN protein was higher when coexpressed with F protein in potato than when expressed on its own in tobacco (0.3 -0.6 µg/g total leaf protein and 0.069 % TSP, respectively) (Berinstein et al, 2005;Hahn et al, 2007). When transiently expressed in N. benthamiana as a pSPhnHN/KDEL, under the control of rubisco small subunit promoter, fused to its own KDEL ER retention signal, recombinant pSPhnHN/KDEL protein accumulated to the highest levels from all of the 5 tested constructs .…”

How can plant genetic engineering contribute to cost-effective fish vaccine development for promoting sustainable aquaculture?

“…Each system has its advantages and limitations and the method of choice is largely depending on what kind of fish vaccines are to be expressed, as briefly described in Table 2. To date, both food and non-food crops (especially tobacco plant) have been used for the development of a number of animal vaccines, such as a poultry vaccine against Newcastle disease (Hahn et al 2007; Yang et al 2007; Li et al 2007; Gómez et al2009; Van Eck and Keen 2009; Wu et al 2009: for reviews see Floss et al 2007 and He et al 2008), rabies (Ashraf et al 2005; Loza-Rubio et al 2008; Roy et al 2010; Loza-Rubio et al 2012), Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine post-weaning diarrhea in piglets (Chen and Liu 2011; Kolotilin et al 2012). The vaccine against Newcastle disease was the first plant-made animal vaccine receiving regulatory approval from the US Department of Agriculture (USDA) Center for Veterinary Biologics in 2006 (www.thepoultrysite.com/poultrynews/8949/usda-issues-license-for-plant-cell-producednewcastle-disease-vaccine-for-chickens; Joensuu et al 2008).…”

Heterologous expression of synthetic chicken IFN-γ in transgenic tobacco plants

“…Plant expression systems have advantages over other in vitro expression systems in terms of low production costs (Giddings et al 2000;Ohya et al 2001;Daniell et al 2001;Leelavathi & Reddy 2003;Chen et al 2004;Arlen et al 2007;Hahn et al 2007). Although expression was lower than in other systems, the cost of producing recombinant proteins in plants could be 10 to 50-fold lower than producing the same protein by Escherichia coli fermentation, depending on the crop (Kusnadi et al 1997).…”