Preparation Of E. Coli Enterotoxin Yolk Antibody And Its Neutralizing Efficiency Against Enterotoxin

Enterotoxigenic Escherichia coli (ETEC, short for Enterotoxigenic E. coli) is one of the mainpathogens causing piglet diarrhea. Among livestock, especially the newborn calves are particularlysusceptible and ill with poor growth, lower production capacity and even death. Undoubtedly itbrings a great loss for animal husbandry. Piglet diarrhea has a close relationship with the bacteriaenterotoxin LT and ST which are produced by ETEC. At present, we mainly take Escherichia colipolyvalent vaccine and drug prevention for the disease. However, the effects are very not ideal.Egg yolk antibody, as a kind of high specific biological agents have significant curative effect inthe prevention and treatment of piglet diarrhea, so this way opens up a new path for the preventionof the disease.We amplified nucleotide sequences whinch the B subunit genetic ltB of heat-labileenterotoxin (LT) and st1a of heat-stable enterotoxin (ST1a) contains18amino acid,connectedtwo sequences via linker and then insered into expression vectors: pET-28a-sumo、pET-32a、PHUE、pCold I、PGEX-6P-1. Linking the ltB gene with HA tag subsequently inserting intopET-28a-sumo as recombination expression vectors with HA tag. After optimizing the expressionconditions, we achived recombinant protein LTA-ST1and LTB-HA. Recombinant proteinLTB-ST1a was used as immunogen to immune8eight-month old HY-LINE brown hens. Immunethree times every two weeks. The immune dose of the first time is0.4mg/ml, inject1mlimmunogen to each hen. The immune dose of the second time is0.6mg/ml, inject1ml immunogento each hen. The immune dose of the last time is0.8mg/ml, inject1ml immunogen to each hen.Collecting eggs on the third day after the second immune then extracting egg yolk antibody bychloroform extraction and using indirect ELISA to detect the antibody’s titer. recombinant proteinLTB-HA was used as envelope antigen to detect the titer of LTB in the egg yolkantibody.accordingly,4ST1a protein where expressed in recombinant bacteria constructed in ourlaboratory was conducted as envelop antigen to detect the titer of ST1a. Results show that inindirect ELISA test, the highest titer of LTB and ST1a is1:9192and1:512. The change trend ofthe two antibodis,titer are nearly consistent. Both of them get the highest titer around the twoimmune, then decrease after the three immune, rise gradually after the third immune. The LTneutralization experiment(rabbit intestines ligation experiment) demonstrate that1:1mix IgY andLT, IgY can neutralize the virulence of LT. If double dilution, IgY can not neutralize the virulence of LT completely. After four fold dilution, IgY doesn’t neutralize the toxins LT. The mixture ofthree different gradient of yolk antibody mix and toxin would increase effusion of intestinalsegments. ST toxin neutralization experiment (Rats lavage experiment) proves that IgY canneutralize the virulence of ST1a and inhibite the emerge of intestinal fluid, beyond thatexperimental mice are healthy living, IW/BW is0.075; While control mice are all dead, IW/BW is0.112.This research confirms IgY which are generated through fusion protein LTB-ST1a infectedhens can neutralize LT and ST1a. Furthermore, the study provides high effective prevention agentsfor piglet diarrhea’s prevention and cure, also supply detection antibody for the further research ofenterotoxin.