Development Of Immunomagnetic Beads-PCR For Listeria Monocytogenes And Preparation Of Single-chain Antibody Against P60 Protein

Listeria monocytogenes is a foodborne pathogenic bacteria,which has the characteristics of wide range of pollution,strong environmental tolerance,high mortality and so on.A series of listerellosis such as meningitis,septicemia,abscess and miscarriage of pregnant women could be caused by Listeria monocytogenes.In recent years,the infection of Listeria monocytogenes has been broken out continually all over the world,which has caused great property loss and casualties.Therefore,it is of great significance to establish a rapid,accurate and reliable method for the detection of Listeria monocytogenes in food.The traditional method of isolation and identification of Listeria monocytogenes has the disadvantages of time-consuming and labor-consuming and can not meet the needs of rapid detection.Immunological methods and molecular biological methods have been widely applied to the rapid detection of pathogens because of their advantages of rapid and high accuracy.In this study,a method for rapid detection of Listeria monocytogenes in food was established by using immunomagnetic beads and duplex PCR.In addition,a single-chain antibody(scFv)against Listeria monocytogenes membrane protein p60 were also prepared.The main results are as follows:(1)The iap gene of Listeria monocytogenes was amplified by PCR,and the amplified fragment was inserted into the pET-28a(+)plasmid,then the plasmid was transferred into E.coli BL21(DE3)to obtain the expression strain.The expressed strains were induced by IPTG to obtain p60 protein,and were purified by Ni-IDA resin to obtain the purified p60protein.(2)New Zealand white rabbit was immunized with p60 protein,and antiserum was collected after four immunizations.The antiserum was purified by Protein G resin,and the polyclonal antibody against p60 was obtained with the titer above 1:12500.Cross-reaction study showed that the polyclonal antibody could recognize Listeria strains with no cross-react of other bacteria.The polyclonal antibody was coupled with carboxyl-modified magnetic beads by carbodiimide method,and the immunomagnetic beads of Listeria strains was obtained.The results of bacterial capture study showed that the capture rate of Listeria monocytogenes reached maximum when the amount of immunomagnetic beads was 20?L and the incubation time was 30 minutes.(3)A duplex PCR method for the detection of Listeria monocytogenes was established by using two pairs of specific primers for Listeria and Listeria monocytogenes and the bacteria captured by immunomagnetic bead as template.Under the optimal conditions,the detection limit of Listeria monocytogenes was 10~6CFU/mL.Smple spiked study results showed that,Listeria monocytogenes artificially contaminated pork samples with 1CFU/g could be successfully detected after enrichment for 8 hours.(4)BALB/C mice were immunized with p60 protein and a phage-display scFv library with a capacity of 1.4×10~7 was constructed.After three rounds of panning and phage monoclonal screening,a phage-sc Fv(P2)which showed specific for Listeria monocytogenes was obtained.Then,the scFv expression vector was constructed,after expression and purification,P2 scFv was obtained.A ELISA based on P2 scFv showed a sensitivity of 10~6CFU/mL to purely cultured Listeria monocytogenes.