Site-directed Mutagenesis, Study On Immunogenicity And Preparation Of Monoclone Antibody For Mutated Listeriolysin O

Listeria monocytogenes, belonging to Listeria genus, is a kind of foodborne pathogenic bacteria which can cause human and animal diseases such as meningitis, encephalitis, sepicaemia, endo-caritis, abortion, abscess and topical purulent damage, and result in the pregnant woman abortion, stillbirth and etc. listeriosis is a serious illness and this is reflected by the apparent high mortality rate in many case, with fatalities averaging approximately 30%. Therefore, it is necessary to develop a rapid detection method for Listeria monocytogenes. LLO is the most important virulence factor which is encoded by the hly gene of L. monocytogenes. In order to increase LLO immunogenicity, prepare high affinity monoclonal antibodies , improve detection sensitivity of Listeria monocytogenes. site-directed mutagenesis to LLO was carried out in this study.In this study, a 1011bp gene fragment encoding hly was amplified by conventional PCR , recombinant plasmid pMD18-T-hly was constructed, site-directed mutagenesis of amino acid residue 52(L52F)of LLO was undertaken, respectively. The target gene hly and mutated gene L52P were subcloned into the expression plasmid pET-28b to construct recombinant prokaryotic expression plasmid pET-28b-hly and pET-28b-L52P respectively. The hly and L52P gene were expressed as inclusion bodies by inducing of IPTG respectively, so purification of LLO and mutated LLO wer carried out by using Ni-NTA affinity chromatography under denaturing conditions. The rabbit polyclonal antibodies of LLO and mutated LLO were prepared by using purified recombinant protein, respectively. LLO antibody titer was 3×10~5, mutated LLO antibody titer was 6×10~5 by ELISA, ELISA results showed that the OD value of mutated LLO antibody is higher than that of LLO 0.44. This shows that immunogenicity of mutated LLO was enhanced, significantly. Monoclonal antibody was prepared by hybridoma technique using purified mutated LLO, 11 positive hybridoma cell lines are gained at first after detecting and screening, but only 2 hybridoma cell lines are left after 3 subcloning. They are named as 2B6 and 5D3, respectively. ELISA results show that ELISA titer of 2B6 is higher than one of 5D3, and the ELISA titer of 2B6 can reach 1:320000. The titer of abdominal fluid is about 533 times more than hybridoma cells supernatant averagely. The preparation of monoclonal antibody of mutated LLO lay a good foundation for research of LLO and rapid diagnostic method for Listeria monocytogenes.