ETEC K99/987P Pili Protein Antibody PPA-ELISA Detection Method Of Genetic Engineering Bacteria And Establishment Of High Density Fermentation Of K88-K99Fushion Protein Research

A PPA-ELISA to detect the anti-K99IgG levels has been developed by using the porcineenterotoxigenic Escherichia coli (ETEC) purified K99fimbrial as coating antigen. ThePPA-ELISA was checked with serum samples obtained from rabbits and pigs, and the optimalparameters affecting the method were also determined as follows: Antigen coated ELISAplates at4℃overnight; The optimal coating concentration of K99,4.94μg/mL; The optimaldilution of corresponding serum samples, rabbits1:50, pigs1:200; The optimal blockingsolution,100g/L skimmed milk powder; The optimal blocking time,0.5h; The optimal serumsamples combine time1h; The optimal PPA concentration gradient,1:2000; The optimal PPAcombine time,0.5h; The optimal substrate reaction time at room temperature,10min; Thepositive-negative cut-off point, in rabbit sera direct OD4500.32, in pig sera direct OD4500.45.A PPA-ELISA to detect the anti-987P IgG levels has been developed by using theporcine enterotoxigenic Escherichia coli (ETEC) purified987P fimbrial as coating antigen.The PPA-ELISA was checked with serum samples obtained from rabbits and pigs, and theoptimal parameters affecting the method were also determined as follows: Antigen coatedELISA plates at4℃overnight; The optimal coating concentration of987P,27.44μg/mL; Theoptimal dilution of corresponding serum samples, rabbits1:10, pigs1:200; The optimalblocking solution,100g/L skimmed milk powder; The optimal blocking time,0.5h; Theoptimal serum samples combine time1.5h; The optimal PPA concentration gradient,1:2000;The optimal PPA combine time,0.5h; The optimal substrate reaction time at roomtemperature,20min; The positive-negative cut-off point, in rabbit sera direct OD4500.37, inpig sera direct OD4500.37.The screening of five medium，set the initial of the culture medium were6,6.5,7and7.5four gradient for protein expression；the optimum initial ph=7of culture medium wasdetermined;the medium of2*YT+0.5%grouse has the characteristics of bacteria count and fast growth;high content of protein ecpression of PH=7,the optimal induction time of2.5h~3h,induced expression for4hours,dissolved oxygen control in30%.