Transformation Of The Genes Encoding The Fmdv Immunogens In Maize

Foot and mouth disease(FMD) is the most serious infectious disease of livestock in the world. Inactivated vaccine to prevent foot and mouth disease is the main measure. With the development of plant genetic engineering, the expression of antigen of transgenic plants could be possible. Relatively traditional vaccine plant is a safer and more economical expression system. In this study FMDV immune genes were integrated into the genome of diploid maize. DNA of maize leaf was extracted . Based on PCR detection of plant DNA, the positive plant was screened and laied the foundation for the development of the plant vaccine.As the technology of the production of plant protein has matured, Making use of the oral vaccine plant turn to be possible. In this study maize embryos callus is selected for the transformation as material. For actual use the FMDV structural protein VP1 genes and the gene P12A-3C were cloned. The plant expression system was optimized. Specific results are as follows:1) The total RNA of virus was extracted from Asia1/HB of sulking mouse virus .Gene P12A and 3C were amplified from viral RNA by RT-PCR and cloned into pGEM-Teasy vector, analysiing P12A and 3C genes with reference, the genes P12A and 3C were successful obtained . Then the restriction sites were introduced by PCR amplification and the plant-expression vector pC1300-P12A-3C was const -ructed. Its transformated into the Agrobacterium and got gene into maize callus by Agroba cterium mediated transformation.2) According to the preference of maize codon, O/HK/99 strain VP1 sequence, Asia1 type FMDV VP1 sequence, E. coli sensitive enterotoxin B subunit (LTB) and Vibrio cholera toxin B subunit (CTB) were optimized. 1095bp Kozak-LTB-linker-Asia/VP1 and 1101bp the Kozak-CTB-linker-O/VP1 genes were Synthesized .Plant bivalent carrier pC1300-LTB-Asia1/VP1-CTB-O/VP1 was Constructed and got into law Through gene gun;3) Maize transformation system was optimized with MS and N6 medium. The maize "White 18" callus materials were selected and grow with four-generation in low temperature. Infection with the activate ed Agrobacterium and gene gun are used in transformation. Maize callus were in the dark cultured with 3d .then with the three concentration gradient selection of Hyg, cultivation and differentation of callus of resistance. The growth of plants is controled with MET and ABT. The selected plants with height of 2.5-3 cm grow stronger in the growth medium. Plants resistance was improved with the regulator.the survival rate is 25%.plants grew well and received the first generation of transgenetical maize seeds; 4) Immune gene VP1 and P12A-3C were turn into maize callus in different ways. PCR detection of genetically modified maize show that FMDV immune gene P12A-3C was integrated into the maize genome.