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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 222-228 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) coding sequences which could also be the reason for these lower expression levels. Purification of rHA and rNA gene in E.coli: The rHA and rNA proteins were cloned in such a way that the expressed gene will have a Nterminus His-tag so as to enable the purification simpler and quicker. The cells expressing rHA and rNA were scaled up in a 2 liter flasks and the rHA and the rNA proteins were purified through Ni-NTA chromatography. The elution fractions were analyzed on SDS-PAGE and confirmed by western blot which showed a 66 kDa rHA and 50kDa rNA protein (Fig. 3). The fractions containing purified proteins were pooled. The purified HA and NA proteins were dialyzed against 50mM sodium phosphate buffer to remove the imidazole and were adjusted to a final concentration of 1mg/ml before storing at -80ºC for further analysis. Fig.3. Purification of rHA and rNA from E.coli. The clones expressing rHA and rNA were induced with 0.5mM IPTG and the cells were harvested 4 hours post induction. The rHA and rNA were purified through Ni-NTA column chromatography. The purified fractions were analyzed in a 12% SDS-PAGE and western blot. M-Marker; 1-SDS-PAGE showing purified rHA; 2-western blot showing purified rHA; 3- SDS-PAGE showing purified rNA; 4-western blot showing purified rNA 226 Haemagglutination assay : The purified rHA protein was adjusted to a final concentration of 1mg/ml and the haemagglutination activity was analyzed using various RBCs. The guinea pig RBC showed the maximum haemagglutination titer followed by chicken and horse. The rHA protein showed a HA titer of 32 with chicken RBC (Fig. 4), 64 with guinea pigs RBC, 4 with horse RBC and no agglutination with sheep RBC. This clearly demonstrates the use of bacterial expressed rHA in diagnostic assays and as a candidate for subunit vaccine. Fig. 4. Haemagglutination activity of purified rHA. The purified rHA protein was adjusted to a final concentration of 1mg/ml and the haemagglutination activity was analyzed using various RBCs. The guinea pig RBC showed the maximum haemagglutination titer followed by chicken and horse. The rHA protein showed a HA titer of 32 with chicken RBC, 64 with guinea pigs RBC, 4 with horse RBC and no agglutination with sheep RBC. Conclusion A variety of reports support the role of rHA and rNA as potential vaccine and diagnostic tools for avian influenza. Although, both HA and NA antigens are known to elicit neutralizing antibody response in animals (15, 16, 17), antibodies to HA is known to play a major role in virus control while antibodies to NA helps in partial not complete protection. Even though HA alone can provide maximum protection in animals from Cloning, Expression and purification of H5N1 in E. coli
Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 222-228 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) the disease (15, 16, 18, 19), few studies have already reported that addition of NA with HA improving the vaccine efficiency (17, 20). Hence, a detailed study on the combination of these two antigens as a vaccine candidate is a necessity. These rHA and rNA can also be used in the diagnosis of avian influenza as there are 16 HA subtypes and 9 NA subtypes have already been reported and for each type the HA and NA antigens can be readily expressed and purified in E.coli with in a very short duration. In addition to the possibility of using as subunit vaccines these antigens expressed in E.coli also pave a way for the development of easier and quicker diagnostic tools for avian influenza. Acknowledgement This study was supported by a funding from Council of Scientific and Industrial Research (CSIR), New Delhi, India. References 1. Lamb, R.A. and Krug, R.M. Orthomyxoviridae: The Viruses and Their Replication. Fields Virology, Lippincott- Raven Publishers. (1996) p1353-1387. 2. Claas, E.C., de Jong, J.C., van Beek, R. and Rimmelzwaan, G.F., Osterhaus, A.D. (1998). Human influenza virus A/ HongKong/156/97 (H5N1) infection. Vaccine. 16: 977-978. 3. Cheung, C.L., Rayner, J.M., Smith, G.J., Wang, P., Naipospos, T.S., Zhang, J., Yuen, K.Y., Webster, R.G., Peiris, J.S., Guan, Y. and Chen, H. (2006). Distribution of amantadine-resistant H5N1 avian influenza variants in Asia. J Infect Dis. 193: 1626- 1629. 4. de Jong, M.D., Tran, T.T., Truong, H.K., Vo, M.H., Smith, G.J., Nguyen, V.C., Bach, V.C., Phan, T.Q., Do, Q.H., Guan, Y., Peiris, J.S., Tran, T.H. and Farrar, J. (2005). Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med. 353: 2667- 2672. Subathra et al 227 5. Qiu, M., Fang, F., Chen, Y., Wang, H., Chen, Q., Chang, H., Wang, F., Zhang, R. and Chen, Z. (2006). Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin- or neuraminidase-expressing DNA in BALB/c mice. Biochem Biophys Res Commun. 343: 1124-1131. 6. Johansson, B.E., Bucher, D.J. and Kilbourne, E.D. (1989). Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virol. 63: 1239- 1246. 7. Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B. and Osterhaus, A.D. (2005). Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol. 79: 2814-2822. 8. Yang, K., He, F., Li, S., Zhang, J., Lin, Q., Chen, Z., Li, Z. and Xia, N. (2009). [Expression of H5N1 avian influenza virus haemagglutinin protein in pichia pastoris by high-density cell fermentation]. Sheng Wu Gong Cheng Xue Bao. 25: 773-778. 9. Saelens, X., Vanlandschoot, P., Martinet, W., Maras, M., Neirynck, S., Contreras, R., Fiers, W. and Jou, W.M. (1999). Protection of mice against a lethal influenza virus challenge after immunization with yeastderived secreted influenza virus hemagglutinin. Eur J Biochem. 260: 166- 175. 10. Martinet, W., Saelens, X., Deroo, T., Neirynck, S., Contreras, R., Min Jou, W. and Fiers, W. (1997). Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase. Eur J Biochem. 247: 332-338.
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