Expression Of The Fusion Gene K88-ST And VP4-ST In Tobacco

Rotavirus and enterotoxigenic Escherichia coli are the main pathogens causing diarrhea of young animals. Now vaccination is the optical way to control the diarrhea, but frequent inoculation with higher dose was needed for the currently available dead vaccine, and the live vaccine could result in pathogen release and environment contamination. Oral vaccines produced by transgenic plants would change the traditional means of production and inoculation of vaccines, and the cost of vaccine production would be reduced greatly. Consequently it is very significant to develop transgenic plant vaccines. The RV VP4 and ETEC K88, ST are the main protective antigens and pathogenic factors of these two pathogen. So it is very significant in theory and practice to developing transgenic plant vaccines that against neonatal diarrhea by using the fused proteins VP4-ST and K88-ST as antigens.At first, the plant expression vector pBLGC was reconstructed to pBin438 that we used in our experiments. In this research, the VP4-ST and K88-ST gene was modified by PCR amplification. The kozak sequence was placed upstream of the start codon of the two fusion genes, which was advantageous for enhancement of the gene expression. The modified gene VP4-ST and K88-ST was insert into The plant expression vector pBin438.then these two plant expression vectors pBin-VP4-ST and pBin-K88-ST were transformed into Agrobacterium EHA105 directly. By using the leaf-disc co-cultivated method . VP4-ST gene and K88-ST gene were transferred into tobacco cell respectively. Transformed shoots were selected on solid medium containing Kanamycin. Integration of these genes into the genome of tobacco plants was confirmed by PCR, Southern Hybridization analyses, and protein was shown being expressed in tobacco by Western blot.