Prokaryotic Expression Of E.Coli O157:H7Flagellin Protein And Preparation Of Monoclonal Antibodies Against The Flagellin Protein

Enterohemorrhagic Escherichia coli (EHEC) O157:H7belonging to the Enterobacteriaceae Escherichelloa. It can cause diarrhea in humans, Hemorrhagic colitis (HC), Hemolyticuremic syndrome (HUS), Thrombotic Thrombocytopenicpurpura (TTP)such as serious diseases,can gut a stranger for a long time in the animal,great harm to the health of human and animal husbandry.The infection has become a popuLar global public health problem.To prevent the disease can not be lazy,and to establish a rapid^accurate and convenient diagnostic tests is also very necessary.H7protein for EHEC O157:H7flagellar protein, determine the serotype of E.coli.Studies show that EHEC O157:H7and colonic mucosa epithelial cells or mucus combined with process occurs in the early stages of an animal in vivo engraftment in bacteria,however flagella during that time to play the role of a physical connection point.It is on the induced natural immune response at the same time can produce the early inflammatory factor,and can induce the body to produce antigen specific immune response.Prepared in a similar manner EHEC O157:H7monoclonal antibody are mostly against bacteria and pathogenic factor antigen monoclonal antibody. Escherichia coli0157contains many bacterial antigens and antigen-H7E. coli flagella. So,H7flagellar fuLl-length protein is an integral part of the immune antibody in the preparation of specificity of detection. The basis of monoclonal antibody technology in recent years has been widely applied in biomedical research.In diagnosis and treatment of diseases,monoclonal antibody become indispensable tool in many researchers to study.In terms of immunology,strong specificity,high sensitivity,and can continuous and unlimited supply is monoclonal antibodies in comparison with the advantages of polyclonal antibody.At present,the monoclonal antibody has widely used in medicine,agricuLture,food,environment and so many fields.This study amplification EHEC O157:H7flagellar protein gene through genetic engineering methods,the size of1755bp.Insert the purpose fragment into expressionvector pET-28a,and build arecombinant expression plasmid pET-28a-Flic. Insert the plasmid into E.coli BL21(DE3),under the condition of37℃. After IPTG induction4h use uLtrasonic cracking bacteria liquid.Through sds-page analysis,the fusion protein can efficiently expression in E.coli BL21(DE3).The size of the fusion protein is66KD same as the expected.And it is soluble expression. The fusion protein was proved to have better immunogenic by immunoblotting techniques,in which anti-His Monoclonal antibodies can combine with the fusion protein specifically.Through the nickel column affinity chromatography purification the fusion protein,and use it immune BALB/c mice. The spleen cells of BALB/c mice immunized were fused with mouse myeloma cells SP2/0. Four hybridoma cells secreting anti-H7protein antibodies were screened by indirect Elisa method, through the His-Flic-H7fusion protein and the His label protein package Enzyme label plate.Get two cell lines which can stability secreting antibodies, by two times limited dilution method. The antibody belongs to IgM subtype.The antibody is determined favorable specificity by western blot and agglutination test. Two cell lines intraperitioneal injection in mice.The ELISA titer of ascites prepared sufficiently are214and213. Preparation of monoclonal antibodies against the E.coli0157:H7flagellin protein lays foundation for the virus specificity, high sensitivity, simple and rapid detection method.