| The aflatoxin B1 is one of the most toxic mycotoxin in the natural world. The international organizations take it as 10, 000 times as basic toxin unit comparing to other materials. The major harms to human lies in its strong performance of the toxic and carcinogenic. It is produced by the fungus Aspergillus flavus, which is a parasite widely existing in peanuts, corn, and other food. In the modern society, the people have to put more and more concern on the issues of the potential risk of food with the economic development,which has attracted the attention of governments world wide. Among all the governments, the EU has been carrying out the most stringent standard of the safety and test on the food since 2000, in which the AFB1 is limited in 2ug/kg and the total aflatoxin is no more than 4ug/kg. It is because of such restrictions which has created new barriers to trade among countries, which also caused the great economic loss of exporting the agricultural goods to other countries in China.Our research begins with the antigen AFB1-BSA, which is made of the couple of the micromolecule and carrier protein, to immunize mice, then assayed the titer of anti-serum by indirect ELISA after the several round immunization of mice and obtained the specific mouse which the most titer of anti-serum reached 64,0000. Then the total RNA of spleen was extracted after the specific one was killed. We have got the targeted genes of VH, VL and Linker, which is used to connected VH with VL in scFv antibody, through the RT-PCR according to the corresponding primers. Then the gene of scFv was amplified through the special PCR according to the designed primers by overlap extension. Then the scFv gene was connected with the phagemid vector pCANTAB-5E, provided by Pharmcia company, after they was digested by the specific enzymes SfiI and NotI, then the ligated product was electro-transformed into E.co//TG1.Finally we have got a scFv antibody library . as large as 108 clones.When the scFv library was growing up to the log-phase, the helper phage M13KO7 was added to the culture. So the scFv antibody was displayed in N-terminate of the bacteriophage protein and the library of phage antibody was prepared. It could be achieved to obtain some specific antibodies of resistance to the antigen AFB1-BSA through the ELISA test after three rounds of panning-concentration-expansion. Then the E.coli strain HB2151 was infected by the supernatant of the bacteriophage and the relative reaction of the antibody secreted in medium or in periplasm was detected by ELISA after the soluble antibody was prepared. We had got three special strains which could secret the soluble proteins of antibody and they were 3C3, 3B3 and 4A2 respectively.The scfv antibody was more widely applied in the treatment disease and...
|
PDF Full Text Request