Prokaryotic Expression Of Listeria Monocytogenes Hly Gene Fragments And Preparation Of Monoclonal Antibody Against Listeriolysin O

Listeria monocytogenes is a gram-positive,facultatively intracellular bacteria, one of theimportant foodborne pathogenic microorganisms. The bacterium could cause severe syndromes，such as encephalitis，meningoencephalitis and septicemia，the case fatality rate was very high,susceptible population mostly and distribution widely.Infection by LM is mediated by a number ofvirulence factors, in which most important is ListeriolysinO(LLO).LLO is a about60kupore-forming toxin, encoding by hly gene, necessary for LM infection into the target cell andescape the phagosome.The hly gene fragments was amplified by PCR according the templete of LM DNA and weamplified1600bp,980bp,675bp,540bp,620bp epitope of the hly gene fragments was constructedinto pMD18-T Vector, after DNA sequencing analysis the hly gene fragments into prokaryoticexpression vector pET30b. The recombint was transformed into E.coli BL21and expressedoptimally. Expression results showed that the all segmented expressed protein is expressed asinclusion bodies, so try the recombinant plasmid molecular chaperone plasmid the PG-KJE8common transformed into BL21, and a final concentration of1mg/ml L-arabinose and0.2mmol/LIPTG inducible expression of the step-by-step. SDS-PAGE electrophoresis analysis showed that theuse of molecular chaperones of the expression system, wherein the recombinant the LLO protein tothe soluble form expression. The application of the soluble form of LLO protein hemolysis wasfound with a certain degree of biological activity and use the LLO positive serum Western blot andELISA analysis found not natural secretion of LLO and LM specifically binding. After culturing theLM culture, SDS-PAGE analysis, exist in60ku size of target protein, and verified by hemolysis testafter the LLO protein, this protein is PEG20000concentrated and inactivated with formaldehyde37°C fixed, to determine the minimum lethal amount of monoclonal antibody prepared byimmunization of mice.The McAbs against LLO were produced by hybridoma technology, the application of indirectELISA method LM supernatant was diluted16-fold package then screen the monoclonal antibody.The positive cells were subcloned three times by limiting dilution.Three strains hybridoma cell linesteadily seceting McAbs of anti-LLO were obtained and were named5E3,3G7,4B7.The isotypes of McAbs secreted by5E3,3G7,4B7were IgG1Western blot detection shows that this type of themonoclonal antibody has a good reaction specificity.It is providing us a great help to make furtherresearch on the pathogenesis of LLO, biological characteristics and the establishment of the fast,specificity and high sensitivity of the detection method of LM provides an important condition.