Clinical Isolation Of Listeria Monocytogenes And Preparation Of FlaA Monoclonal Antibody

Listeria Monocytogenes(LM)is a Gram-positive bacterium that can parasitize in celles.It can infect not only animals,but also humans,causing public health problems.In order to do a good job in the healthy breeding of mutton sheep in Jiangsu province,as well as the risk assessment of mutton-related pathogens,this study conducted a clinical test of L monocytogenes,in major sheep-breeding areas in Jiangsu province,and developed a monoclonal antibody against the flagellin of L,monocytogenes.1036 sheep-anal swab samples were collected from the main sheep-raising areas in Jiangsu from 2017 to 2019 and tested by PCR method,and 2(0.19%)samples were determined to be positive as L.monocytogenes.This result indicates that Listeria.has a low frequency of infection in mutton sheep in Jiangsu.Two positive samples detected by PCR were prepared for bacterial isolation with PALCAM agar,a specific medium,and two strains of L monocytogenes(named JC8 strain and JC46 strain,respectively)were obtained.Through mass spectrometry identification and biochemical analysis of the isolates,JC8 strain and JC46 strain were determined to be L.monocytogenes.The multiplex PCR was used to identify the serotypes of the isolates and the two isolates belonged to 1/2a or 3a.The biofilm formation ability test showed that JC46 strain had weak biofilm formation ability,while JC8 strain had no biofilm formation ability.In order to guide the treatment of L.monocyt.ogenes.infection in Jiangsu province,drug sensitivity experiments were conducted on JC8 strain and JC46 strain The results showed that both strains were sensitive to streptomycin,kanamycin,gentamicin,erythromycin,cefradine,lincomycin,roxithromycin and azithromycin,and were resistant to polymyxin and enrofloxacin;Besides JC8 strain is resistant to penicillin while JC46 strain is sensitive to penicillin.FlaA is a flagella protein with good antigenicity,and its antibodies can be used to detect the bacteria.According to the FlaA gene(flaA)sequence published by GenBank,a pair of specific primers was designed for the amplification of the FlaA structural protein.Target fragment was amplified and inserted into pMD-18-T.Sequence analysis showed that the amplified gene was consistent with the published sequence.The target fragment was excised from the pMD-18-T vector with BamH I and Sam I and cloned into the expression vector pGEX-6P-1;the obtained plasmid pGEX-6P-1-FlaA was transformed into E.coli BL21(DE3).After induced by IPTG,the fusion protein GST-FlaA(about 55 kDa)was expressed in the supernatant and precipitate.A GST purification column was used to purify the protein.Anti-GST serum was used as the first antibody,and goat anti-mouse IgG was used as the second antibody for Western-blot.The results showed that the fusion protein could react with anti-GST serum.Using the obtained purified fusion protein GST-FlaA as immunogen,after immunizing 6-week-old BALB/c mice according to the conventional procedure,the spleen cells were fused with SP2/0 myeloma cells.Positive cell lines were screened by indirect ELISA,and two cell lines(3C4 and 6F8)that could secrete anti-FIaA stably were obtained.The results of antibody subclass identification showed that the antibodies produced by the two strains of cells were both IgG1 and the light chains were ? type.Ascites was obtained by in vivo inducing method.3C4 and 6F8 cells produced monoclonal antibodies with titers of 1:256000 and 1:64000,respectively,and both monoclonal antibody could react positively with FlaA coating antigen and had no cross reaction with other bacteria.It indicates that the prepared monoclonal antibody can be used for the identification of Listeria.isolates.