After washing with PBS 3 x, the cells were subjected to the same incubation with 1.5 pmol of rP104-1-S/Fc followed by FACS analysis, as described above. newborns who are more weak and susceptible to the toxicity [4]. The most promising measure for the protection of humans and animals against infection is vaccination. Vaccination with SAG1, affinity-purified from the RH strain, produced high survival rates and significantly decreased brain cyst loads in mice [5]C[8]. Also, the use of a combination of antigens delivered as plasmids coding for regions of micronemal proteins, including MIC2, MIC3, MIC4, M2AP, and AMA1, resulted in a significant reduction (84%) in the number of cysts [9]. Interestingly, almost all protective molecules seem to be involved in the parasite-host interaction [10]. Thus, the exploration of this type of molecule from appears to be extremely important for vaccine development. has the remarkable ability to invade a broad range of cell types. This parasite is believed to attach to host cells via ubiquitously expressed surface molecules of the host, or each host cell type may carry a unique receptor that is bound by a particular parasite molecule [11]. Fourteen PAN/apple domain proteins have been detected in may mediate inter-specific interactions, thereby providing a link between host and parasite. To explore the function or characters of other members of the family, we selected a sequence containing several PAN/Apple domains from the GenBank, characterized the protein and identified one of its receptors on host cell surface. Glycosaminoglycans (GAGs), or mucopolysaccharides, are long unbranched polysaccharides consisting of a repeating disaccharide unit [18]. GAGs include chondroitin sulfate (CS), dermatan sulfate, keratin sulfate, heparin, heparin sulfate (HS), and hyaluronan, among which CS is the most prevalent GAG component [19]. Cell surface GAGs are utilized as a receptor by a variety of pathogens, including RH Rabbit polyclonal to ADPRHL1 strain [24] were inoculated in a monolayer of Vero cells [24] cultured in Dulbecco’s modified essential medium (DMEM; Nissui, Tokyo, Japan) supplemented with 7.5% fetal bovine serum (FBS). 293T cells [24], [25] were cultured in DMEM with 10% FBS. CHO-K1 cells and two mutant strains of CHO-K1, Sf9 [24], [25] and Tn5 Levamisole hydrochloride [25]) were cultured in Sf-900II SFM (Invitrogen, Carlsbad, CA) and Ex-cell 405 (SAFC Biosciences Inc., Lenexa, KS), respectively. Recombinant protein synthesis Using the sequence obtained from GenBank (“type”:”entrez-protein”,”attrs”:”text”:”CAJ20677″,”term_id”:”95007456″CAJ20677), primers were designed for plasmid construction in pBSV-Fc-8His [25]. The N-terminus of the protein contains Levamisole hydrochloride four repeats of similar amino acid residues; the forward primer, P104-1-Fc-F (RH strain following propagation in Vero cells using Trizol reagent (Invitrogen). Next, RT-PCR was done using the SuperScript III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). The amplified products were cloned into pBSV-Fc-8His and their sequences confirmed. Subsequently, the positive clones were co-transfected with BaculoGold DNA (BD Biosciences, San Jose, CA) into Sf9 insect cells, and used to infect Tn5 cells. The fusion proteins, designated as rP104-1-S/Fc, rP104-1-B/Fc, and rP104-2/Fc, were purified from the lysate of the culture medium of the infected Tn5 cells. Moreover, the expression of Fc-recombinant proteins was confirmed by Western blotting using anti-mouse Fc antibody. Expression of the GST-recombinant protein (rP104-1/GST) in pGEX-6P-2 Levamisole hydrochloride was carried out according to the manufacturer’s protocol. Open in a separate window Figure 1 Analysis of the P104 protein sequence.A. Levamisole hydrochloride The signal peptide is indicated by black rectangle. A1, A3, A5, and A7 are indicated by a dark gray rectangle. A2, A4, A6, and A8 are indicated by a light gray rectangle. A9 and A10 are shown in light black and shiny black rectangles. The arrows at the N- and C-termini indicate the designated primers; the corresponding PCR products are indicated by black bands. B. The putative apple-like structure of a PAN/apple domain. C1CC6 (shown in bold) indicate the six cysteine residues that form three disulfide bridges. Anti-rP104-1/GST serum preparation, Western blotting, and immunofluorescence assay (IFA) Mice were immunized three times with rP104-1/GST to produce anti-rP104-1/GST antibodies. This work was approved by the Research Ethics Review Committee of Graduate School of Agricultural and Life Sciences, the University of Tokyo (Approval no. P08-183). Anti-sera and normal sera were prepared and collected as described previously [28]. For Western blotting, strain RH was propagated and lysed by passing the cells though a #27 syringe and filtered using a 5-m filter. The purified tachyzoites were lysed in 1 SDS-PAGE.