Preparation And Identification Of Monoclonal Antibody Against TSP3of Cryptosporidium Patvum

A number of severe diseases of medical and veterinary importance are caused byparasites of the phylum Apicomplexa.These parasites invade host cells using similarsubcellular structures, organelles and molecular species. Proteins containing one ormore copies of the type I repeat of human platelet thrombospondin (TSP1), are crucialcomponents of both locomotion and invasion machinery. Members of this family havebeen identified in Eimeria tenella, E. maxima,Toxoplasma gondii, Cryptosporidiumparvum and in all Plasmodium species so far analysed.Most of these proteins weremicronemal protein which located in the Apicalcomplex of the parasite.A bioinformatic analysis of the C.parvum genome predicted12additionalproteins, named CpTSP1through CpTSP12, possessing one to multiple copies of theTSP1domain. Among them, TRAP-C1and TSP8have been found to be micronemalprotein and may be related to the adhesion and invasion of the parasite.While thelocation and function of other family members are remaining elusive. In this study,MAcbs were prepared against recombinant CpTSP3in order to localize CpTSP3inthe sporozoite.At first, the gene and structural features of C.parvum TSP3were analysed,andfound that it contained a signal peptide and a transmembrane region. Four TSP1structural domains and two apple domains are harboured in the extracellularregion.TSP3is similar to TRAP-C1in the structure. Two pairs of PCR primers weredesigned to divide its encoding gene for extracellular region into two sections.Thefirst part contains two apple domains and one TSP1domains,while the second partcontains three tandem repeats of the TSP1domain.The PCR products were clonedinto pMD18-T vector, then sequenced and subcloned into pGEX-4T-1for expressionin E.coli BL21.The recombinant proteins were purified by Gluthathione-Sepharose4B.Two kinds of soluble recombinant protein were obtained which named TSP3-1and TSP3-2respectively.Six-week-old Balb/c female mice were injected intraperitoneally with50μg ofthe purified recombinant protein in CFA and boosted3weeks later with incomplete FA. The hybrids produced by fusing immunized mouse-speen cells with the myelomacell line Sp2/0were used to produce Mabs. Mabs were selected for furtherexperiments based on their reactivity to recombinant TSP3, as detemined usingELISA and Western blot assay. After three round screening and cloning, two positivehybridoma cells were successfully obtained. Mabs were purifed from the ascites of themice inoculated by these hibrdoma cells. Both of the MAbs were identified as IgG1subtype by Isotyping assay. Localization of the TSP3in the sporozoite were detectedby indirect immunofluorescence.It was shown that the protein located in the apicalcomplex of the sporozoite.Due to most of proteins located in the apical region of theparasite are involved in the invasion process,we suggested that TSP3maybe relatedto the invasive fuction of the parasites，and maybe another MIC protein.