Acquirement Of K88ac-stâ…¡-lta/ltb Transgenic Alfafa And Detection And Analysis Of Foreign Protein K88ac In Rransgenic Alfalfa

Object: Enterotoxin Escherichia coli is main pathogen causing piglet diarrhea, which has result in tremendous economic loss, especially in our stockbreeding manufacture.Many researches have proved that K88ac pilus and ST, LT enterotoxin are major protective protein against Enterotoxin Escherichia coli. So all of them play a crucial role in vaccinal research. Alfalfa is the highest yielding of the temperate forage legume, noted for its feeding value, and contains no known mammalian toxins, pathogens or allergens. Technology for the drying of alfalfa and storage as cubes is well established. It is important for the development of an edible vaccine, to choose alfalfa as vaccine production systems.Methods: In this study, we optimized and developed the transformation system of alfalfa cultivar Jinhuanghou, based on the establishment regeneration system. It is also studied that the immunogenity and stability of transgenic alfalfa foreign K88ac protein.Result: 1.Establishment of genetic transformation system on the alfalfa. At the base of the agrobacterium medated genetic transformation, we optimized every approach of genetic transformation to the alfalfa. The optimal preculture time of explant was 3 days. The suitable OD600 of engineering bacterial suspension was 0.6. The appropriate concentration of Kan was 30 and 50mg/L during the first and second stages after reinforcing cultivation for 7d. 2. Obtain of genetic transformation plant and identification. It was proved by PCR examination that target gene has been transferred into the alfalfa. It was displayed further by RT-PCR examination that the foreign gene was of a certain expression level in the positive plant. 3. Research of immunogenity and stability of foreign protein. It was proved that foreign protein expressed had immunity by DIBA. The result of ELISA indicated that the immunogenity of foreign protein had little relation to temperature.Conclusion: we optimized every approach of genetic transformation to the alfalfa and constructed its transgenic system. The system was successfully used in acquiring the transgenic K88ac-STâ…¡-LTA2/LTB. The result showed that transgenic plants of alfalfa were gotten and the genes were inserted into genome of alfalfa by PCR,RT-PCR,DIBA and ELISA. Moreover ELISA indicated that mmunogenity of foreign protein had little relation to temperature. It is of great significance for the research of transgenic alfalfa vaccine against ETEC.