Screening Of The Receptors Of Chicken Shigella IpaC Protein Using Immunoprecipitation Method

Shigellosis is caused by Shigella which is a gram-negative germ and may be evoke diarrhoea and dysentery with frequent mucoid bloody stools.The symptoms of shigellosis also include abdominal cramps,tenesmus and fever.Shigella can also infect pigs,monkeys,foxes,cattle,chickens,ducks and other animals expect human.In recent research,shigellosis from chicken can infect different species and ages of chickens especially the chicks,which cause serious effect for poultry industry.In some researchs found that shigellosis from chicken not only can cause cross infection between in poultry.Other studies showed that cross-infection between human and avian can also occur,which is not only damage the development of the poultry industry,but also bring health problems to humans.Shigella can invade intestinal epithelial cells and then proliferate and spread.This part must have two elements.First,specific receptors were required for bacterial attaching the surface of intestinal epithelial cells,which has been proven to be present in basolateral membrane of human intestinal epithelial cells.Second,invasive proteins combining with cell surface receptor were required for Shigella.It mainly include mxi-spa invasive gene which encode type ?secretion system and Ipa(A,B,C,D)genes which encode protein.Invasive protein of Ipa B and IpaC combined into complex.The complex combined with receptors which exist substrate of intestinal epithelial cells and further promoted bacterial invading into cells.The experiments in vitro showed that only recombinant purified IpaC protein was able to restore invasion of Shigella to a certain extent.The results show that Shigella from human can infected chickens and cause illness and death.Some reaserchs indicated that Shigella from chicken have the same strain with IpaC protein from human.But it is unclear whether there is one similar receptors of IpaC for Shigella from chicken.The study is to screen cell receptors of IpaC of intestinal epithelial cells.The results are as follows: 1.Prokaryoti expression of IpaC gene of Shigella from chicken and purification of IpaC proteinThe recombinant expression strain pET32a-IpaC/BL21 and pET32 a /BL21 which were constructed and preserved by the laboratory were cultureed and identificated of PCR.IPTG of 1.0mM was selected as inducer to express.Ultrasonic disruption instrument was used to dissociate bacterial liquid,the supernate was purified with Ni-Agarose His tag.Washing Buffer which containing 50 mM imidazole wash out miscellaneous protein,Elution Buffer which containing 250 mM imidazol wash out interest protein.The purified proteins was identified with SDS-PAGE and Western-blot.The results of SDS-PAGE showed that IpaC and control proteins were sucessfully expressed.The results of Western-blot showed that IpaC and control protein posessed biological activity.2.Primary culture of intestinal epithelial cells from chicken embryo and extraction of membrane proteinsEintestinal tissue was isolated from 18 days age of chicken embryos,then mixed liquor of 300U/mL XI collagenase and 0.1mg/mL I neutral protease were used to digest intestinal tissue.The method which the cells can stick a wall according to different time was used to purify intestinal epithelial cells.The result of alkaline phosphatase staining was positive.Membrane proteins extraction kit was used to extract membrane proteins of intestinal epithelial cells which had been cultured for 48 h.The result of Western blot with Na+/K+-ATPase ?1polyclonal antibody from rabbit showed that membrane proteins of intestinal epithelial cells were extracted.3.Screening receptors of IpaC protein of Shigella from chickenIpaC and carrier proteins were labeled by biotin-labeled protein kit.We took advantage of streptavidin-biotin beads and biotin marker IpaC protein separating with magneticto,as Biotin and avidin system has a strong binding ability.Membrane proteins of intestinal epithelial which can bind with IpaC protein was combinded to the beads.The beads with proteins will be separated by adding the protein sample buffer.We select the target band which have binded with IpaC protein according to SDS-PAGE and Western blot.Screened protein bands were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS)and analysis of bioinformatics.Annexin A2,37 kD Laminin receptor precursor,LIM and SH3 protein 1,ras-related protein Rab-43 initially were candidate receptors of IpaC protein.The amino acid sequences of candidate receptors were cleared,the test about the exactly receptors of IpaC is currently under way.The structure and biological function of the receptor of IpaC would be studied further.