Thus, the HAH5 proteins purified by IC (HAH5IC) or directly from the culture supernatant of transformed CHO cells (HAH5sC) were used to coat ELISA plates in order to evaluate its capacity to bind antibodies induced by the HACD protein purified by IC (HACD IC) or by size exclusion chromatography (SEC) (Fig. 7). For the positive control, wells coated with HACD purified by SEC (HACD SEC) and the sera of chickens immunized with the same protein were used. In the negative control, measures were carried out coating with the protein HACD SEC and using the sera of chickens immunized with PBS. The ELISA assay coated with the protein HAH5IC showed OD values of 0.61 when the
sera of chickens immunized with HACD IC were tested (Fig. 7A), indicating the existence of anti-HAH5 antibodies. The proteins HAH5sC and the one obtained in the supernatant of SiHa cells transduced with a recombinant adenoviral vector (HAH5sS) were buy Tofacitinib also
able to bind antibodies from the chicken sera used in the previous experiment showing OD values of 0.67 and 0.63, respectively (Fig. 7B). More interestingly, the ELISA assay performed with the protein HAH5IC detected anti-HAH5 antibodies in the sera of chicken immunized with the protein HACD SEC, showing an OD value of 0.69 (Fig. 7C). In all cases, positive and negative controls showed OD values around 0.95 and 0.09, respectively. After the emergence of the HPAIV H5N1, selleck chemical poultry and human health have been compromised. Also, it has caused a serious economic trouble owing to the obstruction of poultry trade industry worldwide . The Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (OIE) have made huge efforts to organize accurate strategies for circumventing or diminishing the damages caused by the H5N1 virus. Among
them, a vaccination program together with biosafety measures which include surveillance, quarantine and sanitation are crucial ,  and . Establishing analytical methods for differentiating infected from vaccinated animal (DIVA) Phospholipase D1 and surveillance require a strong platform for protein production, which need a robust and reliable expression system able to produce large amount of protein. In this study, an expression system based on the stable transduction of CHO cells with a recombinant lentiviral vector carrying a synthetic gene with the coding sequence of the HA protein from the HPAIV H5N1 was assessed. The generation of genes by chemical synthesis allows the obtaining of the desired genes in a short period of time, avoids manipulation of strains from HPAIV, the codon usage could be rearranged according to the expression system and allows the addition or removal of regulatory sequences that modulate the expression of the gene of interest. The molecule HA derived from the HPAIV H5N1 A/Viet-Nam/1203/2004 was selected for being lethal to chickens, ducks, ferrets and humans  and .