Browsing by Subject "Recombinant proteins."
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Item New viral vectors for the expression of antigens and antibodies in plants.(2009-06-30T13:49:22Z) Liu, Zun, 1974-; Kearney, Christopher Michel, 1958-; Biology.; Baylor University. Dept. of Biology.Plants viruses are increasingly being examined as alternative recombinant protein expression systems. Future development of plant virus expression vectors needs to focus on the most important economic hosts, namely cereals and legumes, to develop tools to aid breeding of such hosts and systems for edible vaccine production. Sunn hemp mosaic virus (SHMV) is a tobamovirus, which infects leguminous plants. This work reports on new SHMV-based viral vectors for high yield of target proteins in legumes. In the SHEC vector series, the coat protein gene of SHMV was substituted by a reporter gene. In the SHAC vector series, the coat protein was substituted by a reporter gene and the coat protein gene from another tobamovirus, tobacco mild green mosaic virus (TMGMV). Co-agroinoculation of SHEC/GFP with an RNA silencing suppressor resulted in high levels of local GFP expression by 3 days post inoculation. Co-agroinoculation with SHAC/GFP led to systemic fluorescence in 12-19 dpi. Foxtail mosaic virus (FoMV) is a species of the group Potexvirus, which infects cereal plants. A new viral vector series named FECT was constructed by eliminating the triple gene block and coat protein genes, reducing the viral genome by 29%. Interestingly, agroinoculation of the vector alone results in only slight transient expression, whereas co-inoculation with silencing suppressor genes allows for GFP expression of 40% total soluble protein. Full-sized HC and LC components of an anti-langerin IgG, each carried by a separate FECT vector, expressed and folded into immunologically functional antibody upon co-inoculation. This may prove a useful and environmentally safe vector for both transient expression and perhaps transgenic plants. Mountain cedar (Juniperus ashei) pollen causes severe allergies in Texas and the central USA. Jun a 1 is the dominant allergen protein of mountain cedar pollen and would be a good allergen vaccine candidate. Recombinant Jun a 1 was expressed in Nicotiana benthamiana using an agroinoculation-compatible tobacco mosaic virus vector and isolated in good quantity from the apoplast by vacuum infiltration (100 μg/g leaf material). The recombinant protein samples were characterized. Pectate lyase activity was detected from plant extracts, suggesting the cause of severe necrotic reaction in plants.Item The recombinant expression of two pollen allergens using plant-viral and yeast expression systems.(2006-05-09T19:12:08Z) Moehnke, Marcie H.; Kearney, Christopher Michel, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.Allergic disease causes much distress within industrialized areas of the world, affecting approximately a quarter of the population in such areas. Current immunotherapy involves the administration of increasing concentrations of crude allergen extracts over a period of time, in an attempt to switch the individual's allergic response to that of a non-allergic individual. Such therapy is largely ineffective, especially for cedar hypersensitivity where only 30% of individuals respond after two years of weekly injections, and unwanted and sometimes life-threatening side effects can accompany specific immunotherapy. In an effort to increase its efficacy, as well as circumvent these negative side effects, recombinant DNA technology is being used to produce recombinant allergens that will take the place of crude allergen extracts found in immunotherapy injections. A main cause of allergic disease within south central Texas is pollen produced by mountain cedars, Juniperus ashei. In one study, I cloned a particular mountain cedar allergen, Jun a 3, into a tobacco mosaic virus vector under the regulation of a subgenomic promoter. Infectious viral transcripts were inoculated onto Nicotiana benthamiana plants, and recombinant Jun a 3 protein was detected within these plants at 21 days post-inoculation. The recombinant protein was able to bind anti-Jun a 3 IgG antibodies as well as IgE from mountain cedar allergic patient sera. A separate study also demonstrated the successful expression of recombinant Jun a 3, but in a yeast expression system. The Jun a 3 cDNA was cloned into a yeast expression vector and transformed into Pichia pastoris cells for expression. Western blotting and ELISA experiments confirmed the recombinant Jun a 3 produced by the yeast bound anti-Jun a 3 IgG antibodies and IgE from allergic patient sera. A third study utilized the tobacco mosaic virus-based plant expression system to produce the main Italian cypress allergen, Cup s 1, from Cupressus sempervirens. This recombinant allergen behaved very similarly to a native cross-reactive allergen in its binding to IgG antibodies and allergic patient sera.