Antiserum Preparation Of Molecules Associated With EPEC Adherence And Exploration Of The Neutralization To EPEC

Objective: To build prokaryotic expression vector of molecules associated with EPEC adherence which are bfp A, esp A and intimin C300(300 amino acids at carboxyl end of intimin), purify the protein, prepare the antiserum and explore neutralization of antiserum to EPECMethods:1. Three pairs of primers were designed regarding the genome of E2348/69 as template to amplify the gene of bfp A, esp A and intimin C300, the products then were cloned to p MD-19 T, after sequence identification, target gene were subcloned to p ET32 a, then the resulting plasmids were transformed into engineered E.coli BL21.2. With the inducation of IPTG, the target proteins were expressed and checked them with SDS-PAGE and Western-blot.3. The targeted protein were purified with the Ni-column, the rabbits were immunized with the purified protein mixed with equal volume adjuvant, one week later of the last time immune, the antiserum were collected from the blood of the immunized rabbit and the titer was titrated with counter immunoelectrophoresis.4. EPEC were neutralized with different diluted antiserum for one hour before infection and the nonimmunized rabbit serum was regarded as the control, the adhesion of EPEC to Hep-2 cells were observed under microscope to judge the effect of antiserum on EPEC adhesion to Hep-2 cells.Results:1. The prokaryotic expression plasmid p ET32a-bfp A, p ET32a-esp A and p ET32a-intimin C300 were constructed successfully and the corresponding engineered bacteria BL21/p ET32a-bfp, BL21/p ET32a-esp A and BL21/ p ET32a-intimin C300 were built. With the inducton of IPTG, the targeted protein mainly expressed in the soluble form, SDS-PAGE and Western-blot showed molecular weight of expressed product are about 42KD、41KD and 54 KD which are consistent with the expected molecular weight2. High purity of Esp A was purified with Ni-column, but failed to get purified Bfp A and Intimin C300 protein, the purification conditions remains to be further explored.3. Esp A antiserum was collected from the immunized rabbits and the antiserum recognized the EPEC in the experiment of agglutination. The antiserum inhibited EPEC adhere to Hep-2 cells effectively within 16 fold dilutions even if the titer tested with counter immunoelectrophoresis was only 1:2.Conclusion: Prokaryotic expression system were constructed of molecules associated with EPEC adherence, which are bfp A、esp A and intimin C300 and Esp A protein containing histidine tag was purified with the Ni-column. Esp A antiserum was harvested from the immunized rabbit, the antiserum inhibited EPEC adherene to Hep-2 cells effectively, so Esp A is the important neutralization antigen to protect EPEC infection.