Preliminary Study On ELISA Method For Escherichia Coli Antibody Detection And Genetic Subunit Vaccine Against Porcine Edema Disease

Edema disease (ED) is a common cause of illness and death loss in pigs during the first 2 weeks after weaning. The disease is an enterotoxemia caused by Shiga-like toxin Escherichia coli (SLTEC) that colonize the small intestine and produce SLT-IIe. SLT-IIe is responsible for edema disease of pigs. SLT-IIe is a bipartite molecule composed of a single enzymatic intracellularly active A-subunit(SLT-IIeA) and a pentamer of B-subunits (SLT-IIeB). The receptor binding capacity of SLT-IIe is associated with the B subunit. SLT-IIeB has immunological activity to protecte Vero cells from native SLT-IIe.To find out the prevalence of ED in our country and develop safe and effective vaccine to prevent and control ED infection in swine, the ELISA assay for diagonosis of ED and the genetic subunit vaccine against porcine edema disease were studied. The main research included as below:1. Cloning and expressing of SLT-IIeB and SLT-IIe3B genes in Escherichia coli The deletion of leader sequence and transmembrane region of SLT-IIeB gene wasamplified by PCR from a Escherichia coli strain called Ee which was responsible for the edema disease in piglets in Hubei province. PCR product of SLT-IIeB gene was cloned into pGEX-KG vector to generate the recombinant plasmid pGEX-SLT-IIeB. There was not any mutation on SLT-IIeB gene by sequencing. The sequence analysis indicated that the SLT-IIeB gene was about 204bp and encoded 68 amino acids. The homology of nucleotide sequence with other twelve published SLT-IIeB sequences on GenBank was 100%, which indicated that it was relatively conservative. Both expression vectors named pGEX-SLT-IIeB and pGEX-SLT-IIe3B were constructed and were then transformed into E.coli BL21(DE3). GEX-SLT-IIeB and GEX-SLT-IIe3B fusion protein were expressed after induction with IPTG The method contained with inclusion body collection — SKL denaturalization — dialysis re-naturalization was used to purify the two prokaryotic expression products. The fusion proteins were confirmed by SDS-PAGE and specific to standard positive serum of ED, which indicated that the fusion protein possessed immunological activity.2. Development of indirect SLT-IIe3B-ELISA for ED-specific antibodies detectionThe SLT-He3B-enzyme linked immunosorbent assay (SLT-IIe3B-ELISA) based on the recombinant protein which has been purified, denatured, and renatured was developed. The optimal conditions for the ELISA were first established by matrix titration. The amount of SLT-IIe3B protein for coating ELISA plates were determined to be 3.15μg /mL.