| Clonorchiasis is a serious food-borne parasitic zoonosis, which is mainly exhibited ashepatobiliary lesions caused by Chonorchis sinensis. This parasite was reclassified as a group1biological carcinogen in2009. It is mainly distributed in endemic areas of Korea, China,Russia, and Vietnam, Currently it is estimated that more than35million people are infectedworldwide, among which more than15million are in China. Despite researchers at home andabroad have conducted a lot of research about the prevention and treatment of the disease,because Clonorchis sinensis have different developmental stages and the antigensingredients are relatively complex, it is not an easy thing to find a strong specific and sensitiveantigen which is suitable for immunological diagnosis. Therefore, the developmentaldirection for the immunological diagnosis of Clonorchis sinensis is mainly to develop newand high specific antigen or antibody to ameliorate the existing deficiencies of variousimmunological methods, to make it more sensitive, specific, efficient, economical,convenient and safe, and to establish a new type of immunological diagnostic method on theexisting basis.Our lab had successfully established cDNA expression library of Clonorchis sinensisadult and screened out five strong reactive antigen genes using immunological method,sequenced the genes and made use of related molecular biology software for sequenceanalysis. On this basis, we artificially synthesized the five strong reactive antigens, and namedthem CsA1、CsA2、CsA3、CsA4、CsA5, respectively. Due to these antigens are peptides about10amino acids, their molecular weight are small, immunogenicity are weak, so it is necessaryto couple with carrier proteins. Using the negative and positive rabbit serum infectedartificially Clonorchis sinensis to conduct specific identification for three carrier proteins:bovine serum albumin (BSA), chicken ovalbumin (OVA), keyhole limpet hemocyanin (KLH).From the reactive extent, BSA is the best choice for carrier protein. The synthesizedpolypeptide antigens were sent Biology Company to couple with BSA; the SDS-PAGEshowed the products had been successfully coupled. The five coupled antigens wererespectively named No8,9,10,11,12, and immunized them to rabbit every two weeks. Firsttime use Freund’s complete adjuvant, and other times use incomplete Freund’s adjuvant to emulsify antigens completely, after every immunization, we collected serum from rabbit earvein to measure its antibody titer. After the sixth immunization, serum was collected fromrabbit heart, the ELISA test results showed the antibody tilter of No9,10, and10were up to1:2048000, No8and11were1:102400. Using ammonium sulfate precipitation and affinitychromatography for antibody purification, the ELISA result approved that we hadsuccessfully removed the hybrid proteins and antisera against to BSA. Then the Clonorchissinensis adult worm were collected from fresh liver of dogs infected Clonorchis sinensismetacercariae. The five antigens were immunolocalized with indirect immunofluorescencemethod. The results showed that CsA1distributed on oral sucker and ventral sucker ofClonorchis sinensis, CsA2ventral sucker, CsA3oral sucker and ventral sucker, CsA4oralsucker, CsA5oral sucker and ventral sucker.This study had immunolocalized the five antigenic epitopes, due to their specialdistribution; we speculated that these antigens had close relationship with adsorption andmovement of clonorchis sinensis in host. If we can make some study of the contact sites, suchas the oral and ventral sucker, make them not adhere to the wall of bile duct or other ducts, wecan remove these parasite from host to play a role of vaccine. The results of this study providetheoretical foundation and experimental basis for study of biological characteristics ofantigens and laid strong foundations for genetic engineering vaccine development. |
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