parvuminfections in a variety of organisms, novel strategies to improve this antigen production and its specific polyclonal antibodies availability are desired. Following a production strategy from gene to antibody, the study conducted here reveals several advantages of using the H partner to produce polyclonal antibodies. and promising technology for the diagnosis and therapy ofC. parvuminfections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen. Keywords:novel fusion partner, immunogens, free-adjuvant immunization, antibody production,Cryptosporidium, CP12 == Introduction SR9011 == Antibodies are important tools in biomedical research. They allow the identification of new genes, the purification of proteins, and the study of protein properties such as structure, function and localization.1,2Most of these applications use polyclonal antibodies, which are produced in response to multiple epitopes of the same target protein (antigen). Polyclonal antibodies are usually raised against a specific protein by immunising an animal with the target protein in its purified form.1 The most effective way of obtaining the high quantities of antigenic protein required for efficient immunization is by heterologous expression in a host cell. The bacteriaEscherichia coliis one of the most widely used organisms for this purpose, as it is easy to manipulate, has a fast growth rate and is relatively cheap to use.3-8However, it also has its limitations; the recombinant protein it produces is not always correctly folded or sufficiently soluble for use in immunization. The development of alternative strategies for protein production that overcome these drawbacks is therefore highly desirable.5,9,10One such strategy is the gene fusion technology, whereby the gene coding for the protein of interest is fused to a polypeptide chain, known as a fusion partner. Fusion partners can simplify protein purification, improve protein production yield, reduce susceptibility to proteolysis and increase protein solubility.6,11-14They have also been reported to increase protein immunogenicity.1,15-18 Fusion partners, such as SpA,2GST,19BB-SpG,18MBP,17and Trx,20have been used to improve both antigen expression and antibody production. However, these fusion partners have also been shown to have drawbacks. In some cases, the resulting recombinant antigen is not sufficiently soluble or pure.1,19In others, the immune response obtained against fusion antigens is often triggered predominantly by the fusion partner itself, rather than by the target antigen.1,2,19,21And in others still, the fusion partners have shown inadequate immunopotentiating properties to elicit the production of sufficient quantities of the antibodies of interest. In such cases, during immunization, it is necessary to co-administer an adjuvant. Despite being widely used for routine antibody production in animals, adjuvants are associated with non-specific immune responses and can cause several side effects and lesions at the injection site.2,18,21,22Therefore, the use of recombinant fusion partners for protein and antibody production has evidently much room for improvement. This work presents the novel H fusion partner for the recombinant production inE. coliand subsequent adjuvant-free immunization of the 12-kDa recombinant protein, CP12 (GenBank ID: XM625821.1), belonging to the parasiteCryptosporidium parvum (C. parvum). The H partner consists of an 11 residues sequence from the N-terminal part of a calcium-binding protein (Fh8, GenBank ID:AF213970.1) excreted SR9011 and secreted by the adult worm ofFasciola hepatica.23The H sequence was recently suggested as a fusion partner, and it showed to improve protein expression yields inE. coli, though it did not function as solubility tag.24 The utility RH-II/GuB of the H tag as a fusion partner for recombinant antigen and antibody production is here demonstrated by using the CP12 protein, which, as a surface adhesion protein, plays a major role in the diagnosis ofC. parvuminfections in various mammals, including humans.25A higher availability of this antigen and its specific polyclonal antibodies is therefore important for cryptosporidiosis prevention and therapy. In addition, the low molecular weight of the H fusion partner (1 kDa) makes it a particularly attractive option for use in antibody production. == Results == == Expression and purification of CP12 inE. coliusing the H fusion partner == The sequence ofcp12gene cloned into pQE-30 and pQEH vectors (Fig. 1) matches 100% identity with the originalcp12sequence except that it SR9011 lacks the original N-terminal peptide signal and transmembrane region. Figure 1.Amino acid and nucleotide sequences of the HCP12 codifying gene. Results of protein purification were analyzed by SDS-PAGE (Fig. 2), and revealed that both CP12 and HCP12 proteins were obtained at the predicted 10 kDa and 11 kDa, respectively. After being purified, CP12 non fused protein achieved a production yield of 0.40 0.050 mg per liter ofE. coliculture while HCP12 fusion protein achieved.