Cloning, Expression Of PeblA And CadF From Campylobacter Jejuni And Preparation, Purification Of Their Polyclonal Antibodies

Campylobacter jejuni is a type of zoonotic pathogen leading to an acute enteritis and food poisoning, which was listed as the second risk pathogen for human and livestock as Salmonella and Shigellae. Establishment of quick detection method of C. jejuni is a kind of valid measure for preventing and controlling the food-borne diseases.Usually the detection methods for C. jejuni include method of isolation and culturing, biochemical tests, immunological assay and molecular biology methods. Immunological screening method played an important role for the detection of pathogenic microorganisms. However, up to date, the relevant products and technology for the detection of C. jejuni was rare.One of the key steps for establishing fast detection method of C. jejun is to obtain specific antibodies. For that purpose, the following work was performed.Based on the investigation of literatures, the PEB1 and CadF proteins of C. jejuni were used as specific antigens. Reports showed that the gene encoding outer membrane proteins i.e. CadF and PEB1 was conserved and the two proteins were detected in different serotype strains of C. jejuni, indicating both of them could be used as detective antigen.With the aid of gene cloning and expression, two outer membrane proteins i.e. PEB1 and CadF were expressed, and then purified by using nickel ions affinity column. By immunization of PEB1 and CadF in Zealand long-eared rabbit, two kinds of polyclonel antibodies against PEB1 and CadF were prepared successfully, with the titer of 1:1024000 and 1:128000, respectively.Dot blot hybridization was used for the evaluation of cross reaction between antisera and test strains. It was found that cross reaction existed between PEB1 antisera and Pseudomonas aeruginosa, Staphylococcus aureus, Shigella flexneri and Lactobacillus bulgaricus, and between CadF antisera and Proteus vulgaris, Vibrio parahemolyticus, Listeria monocytogenes, Staphylococcus aureus and Bifidobacterium breve. Thereafter, by using octylic acid-ammonium sulfate precipitation, impurity proteins was removed from antisera, and furthermore, magnetic beads method was used to eliminate the cross reaction of two antiserum with the above microbes. Finally the purified and highly specific pAbs were obtained.Our reaseach provided a basis and the relevant experimental materials for setting up an immunological method for the detection of C.jejuni.