Monoclonal Antibody Preparation Of IpaC Protein From Chicken Shigella

Shigella is one of the pathogens which cause the human and livestock infected with the bacterial diarrhea. With the deeply study of Shigella, many domestic and overseas reports indicated that Shigella can not only infect the livestock such as pigs,chickens, ducks, but also infected monkeys, foxes, and then caused the severe diarrhea, even the death. Now, Chicken shigellosis has been an emerging infectious disease, it can cause chicken bloody stool, diarrhea, and seriously can cause death.The chicken that had drug tolerance can lead to growth retardation and emaciation,which can cause a great economic loss to the breeding industry. Chicken shigellosis infection can not only occur in poultry between, and may be cross-infection between human and livestock, which result a huge public health and safety risk to human. So far, the infectious mechanism of Chicken Shigella lacks sufficient knowledge and understanding, which affects the control of the disease. Therefore, more in-depth understanding of the mechanism is extremely urgent.Ipa C protein monoclonal antibodies have been obtained using cell fusion measure in this study, which leading the way to the detection of the protein and the interaction between the protein and chick intestine epithelia cells. Simultaneously, it provides a reference on how to estimate the importance of Chicken Shigella in public health. The study researches are as follows:1. The preparation of Chicken Shigella Ipa C protein In this study, expanding culture, inducible expression and nickel affinity purification column have been used to obtain the Ipac protein from the expression bacteria of p ET32a-Ipa C/BL21(DE3) that provided by Henan Agricultural University microbiology laboratory. After SDS-PAGE and Western-blot analysis, it proved that the purified Ipa C protein was transcribed and translated by the Iap C gene from p ET32a-Ipac/BL21(DE3) expressed bacteria.2. Preparation of Chicken Shigella Ipa C immunogen The purified Ipa C protein was dialyzed and then emulsified with isometric Freund’s adjuvant. After that, the immunogen was prepared. The Ipa C protein polyclonal antibodies were acquired by immunizing 6-8 weeks SPF mice. It also provided the experimental material for SP2/0 myeloma cells. Using indirect and indirect competition ELISA methods, it tested Ipa C antigen has good immunogenicity.3. Preparation of Chicken Shigella Ipa C protein monoclonal antibodies The mice which can produce good immune stimulation to Ipa C protein were chosen for cell fusion, and then screened the positive cell, prepared ascites, purified ascites.So the high specificity monoclonal antibodies against Ipa C protein were obtained. The monoclonal antibody was detected using the monoclonal antibody subtype test, and the result was Ig G1. Using ELISA and Western-blot detection, the antibody was specific.4. Identification of Ipa C protein receptor on intestinal epithelial cell of chicken Identification of Ipa C protein receptor on intestinal epithelial cell is based ofmonoclonal antibodies against Ipa C protein. Membrane proteins is extracted from Intestinal epithelial cell of chicken, then refer to virus overlay protein binding assay,Ipa C protein receptor on intestinal epithelial cell of chicken is about 55 K da...