Technical Research Of Bacteriophage Endolysin In Listeria Bacteria Rapid Test

Listeria monocytogenes is a kind of wide distribution food borne pathogens,it always lives in the food and dangers to the aged.Phage endolysin is a general of a kind of efficient cracking bacterial cell walls hydrolytic enzymes by phage gene encoding.It can crack the host cell's cell-wall on the multiplication anaphase,and then release progeny phages.Phage endolysin alsways consist of two domain,they are Enzymatically Active Domain(EAD)and Cell-wall Binding Domain(CBD).CBD decide the recognize specifically.Listeria monocytogenes phage endolysin's CBD has a good specificity identfy to Listeria and different serotype bacterial strain.The passage build a expression vector include CBD gene,extract the target protein,apply on the colloidal gold immune strip to finish the fast test of Listeria monocytogenes.Firstly,amplifi CBDP40 gene inserte into vector p ET,complete the recombinant vecor;Secondly,the recombinant cells were induced with IPTG,extract the target recombinant protein,affinity protein accroding to affinity chromatography technologyand the recombinant protein was analysed for its activity.Finish the preparation of colloidal gold and probe marked,find the most optimal conditions of the way of making colloidal gold and probe marked.Finally finish the consist of colloidal gold strip.Testing the strip's stability,repetitive and specificity.The main results are as follows:(1)Received the restructuring of the recombinant plasmid containing the purpose gene engineering bacteria successful.Using temperature gradient PCR amplification phage lyase gene CBD,with double enzyme digestion and connection,successfully get recombinant plasmid,joined His tags at the same time,its expression in the engineering bacteria.Sent to sequencing reagent company,the purpose gene homology of 100%.(2)Successfully obtained with single increase listeria CBD with biological activities of specific binding of recombinant protein.By IPTG induction,the purpose of the ultrasonic cell crushing to obtain recombinant protein,further using His label use nickel ion affinity chromatography column for purification of recombinant proteins,protein concentration can reach the recombinant protein CBDP40 concentration of 0.908 mg/m L,CBD500 concentration of 0.624 mg/m L.ELISA and Western blot test results are positive.(3)Successful completion of the single increase of listeria monoclonal antibody colloidal gold probe.By optimizing choice heating magnetic stirring,colloidal gold prepared by sodium citrate reduction method,preparation of absorption wavelength of 520 nm,particle size is about 16 nm,the quality good colloidal gold probe.Optimized and selected p H 8.2,protein content mark including 12 g/m L is the best condition,labeled probe of listeria specificity recognition,with biological activity.(4)Step by step,through screening test conditions,the optimal portfolio of all the parts of the strip type test,the successful preparation of a good colloidal gold strip quality.Select the best lines to C concentration of 0.8 mg/m L,T concentration of 1 mg/m L,best sample diluent,preparation of a stable,of the colloidal gold strip for optimal color.At the same time will early experiment preparation of recombinant proteins in CBD for the colloidal gold strip line,realized the single increase fast detection of listeria.