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Cloning and Expression YcbR Gene of Escherichia Coli O157and Antiserum Preparation of Expressed Products

Author: CuiQiang
Tutor: ZhouZhiJiang
School: Tianjin University
Course: Of Food Science
Keywords: E.coli O157 Adherence ycbR Expression Purification Immunization
CLC: TS201.3
Type: Master's thesis
Year: 2012
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Abstract


Escherichia coli O157is a kind of infectious pathogenic bacterium whichthreatens food safety. One of the important pathogenic actions of E.coli O157is theability to adhere to host intestinal cells, but the recent understanding to the adherencephenomena is not comprehensive. Our laboratory found that ycbR gene of E.coliO157is related to the adherence phenomena with HEp-2cells. This research aims atcloning and expressing ycbR gene, and then immunizing animals using expressionproteins to get antibody serum with enough valence, which could be used to do vitroadhesion blockage experiment.This research designed expression primers with restriction enzyme Nco I and XhoI cutting sites according to sequence of ycbR gene. ycbR gene was successfully clonedfrom genome of E.coli O157, which has a high conservation without mutation bycomparing with other reported sequences in GenBank. The target gene was connectedto T vector, and than transferred into DH5α for PCR assay, enzyme cutting assay andsequencing assay. Meanwhile，The physicochemical property and hydrophobic naturewere predicted. The T-ycbR positive recombinant plasmid and pET-28α were cut byrestriction enzyme Nco I and Xho I，and then they were connected and transferred intothe expression host(E.coli BL21). The prokaryotic expression systems of ycbR genewere constructed. The recombinant proteins with His-tag were purified byNickel-column separation and purification after fermentation culture and inducedexpression, which occupied approximately56%of the total bacterial proteins.Purified expression proteins were subcutaneously injected to rabbits asimmunization，followed by cardiac blood collection. Serum of YCBR proteins wasseparated from blood. The valence and sensitivity of antibody in serum was measuredthough indirect ELISA method. Under the antigen consistency of0.5μg/mL、2μg/mL、5μg/mL，the corresponding valence is1:800，1:800，1:1600.The s ensitivity of serum antibody to antigen protein is0.25μg/mL。This research cloned and expressed E.coli O157ycbR gene. Vitro highly efficientexpression was implemented and the serum of expression protein was prepared. Itbuild a foundation to clarify whether the expression products of ycbR gene is involvedin E.coli O157adherence, and then provide E.coli O157inspection in food withscientific basis.

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