The Interaction Between Trichinella Spiralis Antigen And Toll Like Receptor, And Establishment Of Double-antibody Sandwich ELISA

Excretory-secretory (ES) antigen of Trichinella spiralis, i.e. excretion/secretion antigen, is akind of antigenic substances that is excreted/secreted by the worm, is exposed to the immune systemof the host, and then induced the immune responses of the host. Can be used for the study of diseasediagnostics、vaccines and immune mechanisms.Toll-like receptor serves as the pattern-recognitionreceptors. It can recognize pathogen-associated molecular patterns (PAMPs) of the pathogens, andalso calls ligand or agonist of Toll-like receptor. They constitute the first line of defense for the body’sresistance to infection, and play an important role in inherent immunity by recognizing many kinds ofantigens, such as endogenous HSPs and exogenous antigens. HSP70is a kind of the mostconservative HSP, as well as the immunodominant antigen for most antigen infection.In this study, ES antigen of larva-in-muscle was produced by culture method of Trichinellaspiralis in vitro, and used for the immunization of BALB/c mice. And the McAbs(monoclonalantibodies)against Trichinella spiralis ES antigen were screened by hybridoma technology andindirect ELISA.After more than three times cloning, two hybridoma cell strains were successfullyconstructed stably secreting anti-ES McAb.this cell belonging to IgG subtype.The A11McAb titersof ascites were1:2.1×105.The concentration of monoclonal antibody is3.8mg/ml. Western-blot assaydemonstrated that A11McAbs prepared in the context could recognize43、49、53kDa protein.Preparation of rabbit for polyclonal antibodies to set up Double-antibody sandwichELISA.Purified serum titer:1:1.2×106.The concentration of purified polyclonal is8.8mg/ml.Thepurified monoclonal antibody and polyclonal antibody were used as basic detection reagents tosearch for Double-antibody sandwich ELISA for detecting virus.The best working concentration ofpurified monoclonal antibody is7.6μl/ml(1：500)，and polyclonal antibody is4.4μl/ml(1：2000)by thesquare.By the method mentioned above.had no reactive capability with Schistosoma japonicumKatsurada,Toxoplasma gondii, Trichuris suis, Cysticercuscellu-losae, Ascaris suum, Clonorchissinensis, Haemonchus contortus and Paramphistomum. The detection system for the minimum detectable amount of12ng/ml. The minimum detectable amount of Trichinella spiralis culture fluid:5larva/ml. The minimum detectable amount of Trichinella spiralis Ultrasonic liquid:0.25larva/ml.Thefounded sandwich ELISA methods can detect circulation antigen after7days of the Trichinellaspiralis-infection.ES antigen of larva-in-muscle of Trichinella spiralis was purifed by combining preparedmonoclonal antibody and immunoaffinity chromatography. SDS-PAGE analysis indicated that threeprotein bands existed with the molecular weight of43,49and53kDa with the Hybrid proteinconcentration of0.34mg/ml.Based on the accessed TLR2and TLR4sequences in GenBank, Toll-like receptor2(mTLR2)and receptor4(mTLR4) were amplified by RT-PCR from mouse macrophages to obtain the full CDSof the genes. After the sequencing, eukaryotic expression plasmid pcDNA3.1-mTLR2andpcDNA3.1-mTLR4were constructed. The recombinant plasmids were transiently transfected intoHEK293T cell. RT-PCR and immunofluorescence were used to detect its expression and cell location.The results indicated that mTLR2can be successfully expressed and locate the cell membrane aftertransfection. The TLR2activator (pam3csk4) and TLR4activator (LPS) were used to stimulate theHEK293T transfected by recombinant plasmids. The dual luciferase reporter gene system wasemployed to analyze the transcriptional activity of downstream transcription factor NF-κB. Theresults indicated that the luciferase activities of TLR2and TLR4after stimulation were significantlyhigher that the normal saline control group, suggesting that the expressed mTLR2and mTLR4hadthe function of wide-type molecular.Three kinds of antigens of Trichinella spiralis (HSP70, ES antigen of Trichinella spiralis, and43,49and53kDa Hybrid proteins) was added into the HEK293T cell transfected by mTLR2or mTLR4It showed that ES antigen of Trichinella spiralis and43,49and53kDa Hybrid proteins bothinteracted with mTLR2and mTLR4receptors to activate downstream NF-κB pathway. HSP70canonly activate the downstream NF-κB pathway of mTLR2receptor.