|Perez, D.M. - UNIV. NEBRASKA|
|Brayfield, B.P. - UNIV. NEBRASKA|
|Phiri, S. - UNIV. NEBRASKA|
|Woods, C. - UNIV. NEBRASKA|
|Morris, T.J. - UNIV. NEBRASKA|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 29, 2004
Publication Date: August 25, 2004
Citation: Perez, D., Brayfield, B., Phiri, S., Borca, M.V., Woods, C., Morris, T. 2004. Preserved antigenicity of hiv-1 p24 produced and purified in high yields from plants inoculated with tobacco mosaic virus (tmv)-derived vector.. Journal of Virological Methods. 121 (2004) 201-208. Interpretive Summary: The report describes the use of TMV as an expression vector for the expression of foreign recombinant antigens in plants, the HIV-1 structural protein p24. The results showed the improvement of our previously reported TMV system regarding the addition of a poly-His tail which improves 10 times the recovering of the protein expressed. This method is a interesting alternative for the production of p24 for diagnostic purposes.
Technical Abstract: We have previously reported the use of a tobacco mosaic virus-based vector (TMV-30B) for production of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana. We now report the development of the TMV-30B.HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed. Coding sequences from the FMDV VP1 protein and the core protein p24 from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VP1-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VP1 vector, p24-His yields were 10 to 15 times higher than those of VP1-His. Twenty-seven grams of TMV-p24.HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24. Animals developed a strong and specific humoral response to the p24-HIS after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His probed its efficiency by confirming the serology of 50 samples previously tested by, two rapid HIV-1 tests thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world.