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The Preparation And Location Of Monoclonal Antibodies Against The LCCL—domain In Cryptosporidium Pravum

Posted on:2014-07-08Degree:MasterType:Thesis
Country:ChinaCandidate:L C NingFull Text:PDF
GTID:2253330425984956Subject:Clinical Veterinary Medicine
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Cryptosporidium parvum belongs to protozoa intestinal parasites, and it can spread by contaminated food, water, or the dust, etc. The most common symptoms of cryptosporidiosis infection of human are diarrhea, and the disease has been listed as one of the6most common kinds of diarrheal diseases. In1986, the world health organization (WHO) has listed human cryptosporidiosis as a suspect of indicators of AIDS (AIDS). Cryptosporidium parvum is mainly parasitic on gastrointestinal mucosal epithelial cells in various vertebrate, causing intestinal epithelial cell membrane invagination, making the worm parasite parasitize in the intestinal membrane outside the cytoplasm containing worm cavity, and evading the immune response of body, so it is difficult to achieve effective parts with drug treatment.In recent years, with the determination of p. falciparum genome sequence to complete, some studies found many genome of plasmodium falciparum which caused human malaria, and predicted the protein products of genes, which found that the protein products contained at least one LCCL structure domain structure. The homologue of genes caused human malaria parasite was also found in apicomplexan parasites, including the cryptosporidium. This experiment conducted to prepare the tiny cryptosporidium with monoclonal antibody containing LCCL structure domain and positioning the proteins which contained LCCL structure domain with immunofluorescence, then laid a foundation for the further study of Cryptosporidium parvum invasion mechanism.This study randomly selected two Cryptosporidium parvum LCCL-domain protein gene, using DNA extracted from bovine source Cryptosporidium parvum as a template to amplify gene containing LCCL-omain, and picking a positive recombinant to carry on sequencing analysis after connecting with pMD18-T carrier, the results were successfully compared with LCCL-domain gene sequences. Clone the LCCL-domain into prokaryotic expression vector pGEX-4T-1by using gene recombination technology. Recombinant plasmid went by enzyme digestion and sequencing identification transferred into E.coli BL21, and IPTG induced target protein expression, then carried on the analysis with SDS-PAGE and Western blot. Finally recombinant proteins in E.coli which were efficiently expressed were got, and they all had reactogenicity. Immune the BALB/c mice with the mixture of purified LCCL-domain protein and the Freund’s adjuvant, and detect the antibody titer of the immune serum in mice by the method of indirect ELISA. Seclect the mice with high antibody titer, take its spleen cell to fuse with SP2/0cells, screen with the positive hole of indirect ELISA detect antibody titer for three times, and the hybridoma cell lines of monoclonal antibody of two resistant LCCL-domain protein which were stable secretion were got respectively. Using protein A affinity chromatography to purify the monoclonal antibody, and the antibodies titer was above1:106, so identified the class was IgGl. The analysis showed that all monoclonal antibody could detect antigen specificity identification by Western blot analysis. Using the monoclonal antibody of anti LCCL-domain protein as the first antibody, localizating domain proteins containing LCCL-domain by immunofluorescence technology, and the results showed that the sporozoite surface film appeared diffuse fluorescence, which suggested LCCL-domain could be expressed in sporozoite protein.
Keywords/Search Tags:Cryptosporidium parvum, LCCL-domain, prokaryotic expression, monoclonal antibody, immunofluorescence
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