Tobacco Mosaic Virus As A Carrier To The Expression Of Exogenous Peptides In Plants

Tobacco mosaic virus (TMV) is a rod-shaped plant virus with asingle, positive-sense RNA genome of 6395 nucleotides. One of itsseparate 3' coterminal subgenomic mRNAs encodes the 17.5 kDa coatprotein (CP) of 158 amino acids that accumulates to high levels in manyhost plants. Generally, while a small foreign peptide is inserted afterGly155 of the TMV CP, the recombinant TMV can still be assembled andreplicated with high efficiency in host plants. So our attentions arefocused on expressing foreign epitopes fused to CP using TMV-basedvector in tobacco plant.Based on the mutaion analysis of recombinant tobacco mosaicvirus (TMV) particles, which carrys an immunogenic dominant epitope of20 amino acids (aa) from foot–and-mouth disease virus (FMDV) serotypeO1K by using sodium nitrite, a recombinant TMV vector was screened forproducing stable recombinant virus particles and expressing efficientlycoat protein (CP) fusion containing the FMDV epitopes of 20 or 25 aa bydeleting the C-terminal 4 aa of the TMV CP. Moreover, two novel insertedsites in the TMV CP gene were tested to screen more efficient TMVvectors for expressing foreign epitopes. These data show that deletingthe C-terminal T-S-G-P-A-T sequence of the TMV CP effects hardly on,and besides, may be more effective for infectivity of recombinant TMV intobacco plant. Moreover, the favorable immunogenicity and protectiveefficacy of the recombinant TMV particles suggest that the novelrecombinant TMV vectors can be utilized in animal or human vaccinedevelopment.In the analysis of the recombinant TMVF28TGA, in which a foreignpeptide of 28 aa followed by a stop codon of "TGA" was insertedbetween Trp152 and Thr153 of the TMV CP, it was found that the foreignpeptide containing a Cys residue induced a series of mutations inrecombinant viral genome, and a mutant virus (TMVF18TAA) particleswith irregular shape and distorted central channel in the infected tissueextracts. Subsequent mutation analysis of the resulting TMVF28YTGAderived from TMVF28TGA by changing the Cys residue of the foreignpeptide to Tyr suggest that foreign peptide containing Cys residue mightaffect the conformation changes of the recombinant TMV particles andsuccessfully expression of CP fusion in tobacco plants.