Preparation Of Monoclonal Antibody And The Localization Of TPO18 Antigen

Cysticercoids pisiformis is a serious parasitic disease beacouse of the larva of Taenia pisiformis-Cysticercus pisiformis.On the basis of previous work,the specific antigen of oncosphere of Taenia pisiformis –TPO18 was selected and prepare monoclonal antibody and polyclonal antibody by it.Its localization at the Taenia pisiformis was studied to expound the origin.These will further definite the relations between the host protective target and the invasiveness and resistivity.1 Using artificially infected test of Taenia pisiformis,this paper established Cysticercus pisiformis infection model.in dog's body,average survival rate of adult is 92%.While the infection rate of Cysticercus pisiformisis in rabbits is 12.03%.The findings explored Cysticercus pisiformis pathogenesis and laid the foundation for immune prevention of Cysticercus pisiformis.2 TPO18 recombinant plasmid was highly effective expressed with IPTG induction.After purification with chromatography the express recombinant protein was analyzed with SDS-PAGE,which show that the molecular weight of the recombinant antigens for 38 ku,and the size is as same as expected.With the positive serum from natural infection Cysticercus pisiformis to be Western-blot test,which proves that the antigen has a good response.The TPO18 recombinant protein was disposed in different environment to analyze its stability.3 The polyclonal antibody was prepared by immunized the New Zealand rabbit(Oryctolagus cuniculus),and its immunogenicity was detected by Western-blot.The results indicated that the rabbit polyclonal antibody has good reactionogenicity,which can be used to the follow-up experiment.4 The purified TPO18 antigen was used to immunize BALB/C mice with Ferunds complete and incomplete adjuvant.After immunizate,the SP2/0 cell fusion was performed.After screening with TPO18 antigen and GST double system indirect ELISA,20 positive hybridoma cell were obtained.Three of them were further sub cloned for three times with limited dilution method and at last three monoclonal cell lines were gained.it is succeed to gain the monoclonal antibody of TPO18 by inoculating monoclonal cell in enterocoelia of BALB/c mice,and.The results indicated that the titer of ascites is 1?51 200,the antibody belong to the Ig G1 class and light chain wask type.is Kappa;The stability of the ascites was tested and chromosome type of the hybridoma cell was performed,which proved that the monoclonal antibody could satisfy the request of the conventional experiment.5.The ABC assay and the electron microscope colloid gold technique with the mice Anti-TPO18 polyclonal antibody and the monoclonal antibody were applied in the paraffin section histochemistry localization of the scolex,the neck segment and the body segment of adult Taenia pisiformis and the porcine cysticercus.The results indicated that the signal of poly antibody is chearer by monoclonal antibody;after antigen localization of the the porcine cysticercus,the main gathering place of the signal of monoclonal antibody is capsule wall and fibrous layer of the lower of capsule wall,the signal of monoclonal antibody also located in kooklet and chuck membrane;the main gathering place of the signal of poly antibody is in the cell of germinal layer and tapetum;after antigen localization of the the Taenia pisiformis,the main gathering place of the signal of poly antibody is in and around sucking disc,the most obvious signal was found in sucking disc and in the epithelial cell of collecting vessels;the main gathering place of the signal of monoclonal antibody is uterine wall and in the fold of the uterus.