Preparation Of The Egg With Broadly Protection Against Diarrhea Caused By Enterotoxigenic Escherichia Coli

Enterotoxigenic Escherichia coli(ETEC)are major pathogens of diarrhea in infants and young children in developing countries as well as traveler's diarrhea in persons who travel to these areas.With the emergence of antibiotic resistance,antibiotics in traditional clinical prevention and treatment are getting less effective.Passive immunization by oral administration of egg yolk antibodies has been studied extensively in humans and animals' diseases due to its safety?no drug resistance and easily preparation.Adherence of ETEC to the intestine surfaces mediated by adhesins is a critical step in the pathgenesis.It is a hotspot of research about How to block the adhesion and then prevent the occurrence of diarrhea in recent years.Recent studies have found that two nonfimbriae adhesions which called flagella protein(FliC)and EtpA,and EtpA mediates adhesion between flagella and epithelial cells.Both proteins play an important role in intestinal colonization of ETEC,and this novel model and EtpA-like molecules in other motile Gram-negtive pathogens for bacterial adhesion may be function in a similar fashion.In this study we obtained FliC and EtpA proteins through constructing prokaryotic expression vector,and got specific egg yolk antibody after immunized hens.Finally did some research on properties of IgY in vitro,and successfully develop murine model of ETEC intestinal colonization to test the effect of inhibiting colonization.These studies lay the essential groundwork for subsequent development and application of broadly protective egg yolk antibody against ETEC in future.The results were as follows:1.Human ETEC standard strain H10407 was used as materials.After amplificated with PCR,the genes etpA and fliC which play a key role in the adhesion between bacterial and epithelial cells were cloned into the expression vectors,then we successfully constructed a fusion-genes expression plasmid,one of which linked with a hydrophobic peptide(Gly4Ser3),and the linked fliC-fliC taked advantage of the properties of isocaudamer.Through optimizing expression condition in BL21(DE3),the results showed when temperature were 37?,cultivated on shaking-table until OD600 increased 0.5-1.0,added IPTG to a appropriate concentration(2FliC:0.1mM,EtpA:0.1mM,EF:0.5mM),and then continue cultivating for 3-4h,collecting the organism through centrifuging,we prepared a large amount of target protein.2.20 weeks-old Lohmann layer were immunized with 1 mg protein per chicken for three times.Purified specific immunoglobulin of high titer by method of water dilution and salt precipitation.The specificity and titer changing of egg yolk antibody were tested with western-blot and indirect ELISA during trial.Results showed that titers of antibody were 16000?64000 and 64000 for 2FliC?EtpA and EF respectively until 7 weeks after second immunization,IgY produced in the group of Fusion protein could reacted with two kinds of proteins independently,shows that fusion protein induced antibodies against both of them.3.In the test of stability of the egg yolk antibody,when the temperature was below 70? or pH>3,IgY properties kept stable;When the temperature exceeds 70?,IgY losing 80%activity after 5 min;Treated in pH=2 PBS after 0.5 h,IgY titer was negative.while hydrolysis accelerated incubated in the pH=1.2 pepsin,but after trypsin treated,almost no impact on IgY.4.Through the developing a murine model of ETEC intestinal colonization,three common serotype strains were applicated in mouse challenge studies.After challenging,mice were gavaged with egg yolk antibody prepared in experiment 2,and has a different effect on inhibiting the number of ETEC colonized in intestinal,which groups EtpA and 2F after challenged with standard strains H10407 have significantly inhibited colonization(P<0.05)compared with controls,and the group fusion protein has reduced the number of infected mice.E519 and 44815 which do not produce EtpA,but after gavaged with E519,groups 2F and the fusion protein significantly inhibit bacterial adhesion(P = 0.01),while infected with 44815,antibody groups only has the tendency of inhibiting colonization(P=0.09),especially fusion protein EF yolk antibody group.To sum up,EtpA and FliC with prokaryotic expression could be used as immunogen in IgY technology,and after tested in the intestinal colonization of mice,the results showed IgY afford broadly significant protection against ETEC colonization,suggested that IgY has further application in passive immunotherapy of ETEC diarrhea.