Nueraminidase Influenza Research

“EXPRESSION, PURIFICATION AND QUANTIFICATION OF NEURAMINIDASE GENE OF INFLUENZA” A dissertation submitted to the VIT University in partial fulfillment of the requirement for the award of the degree of

MASTER OF SCIENCE In BIOTECHNOLOGY SUBMITTED BY A. SRIKANTH
Register No: 08MSBOO1

UNIVERSITY
(Estd. u/s 3 of UGC Act 1956)

VIT

SCHOOL OF BIO SCIENCES AND TECHNOLOGY VIT UNIVERSITY VELLORE-632014 MAY 2010

DECLARATION
I here by declare that the project work entitled “EXPRESSION,PURIFICATION AND CHARACTERIZATION OF NEURAMINIDASE GENE OF INFLUENZA” submitted in partial fulfillment of the requirement for the Master of Science in Biotechnology from School of Biotechnology Chemical and Biomedical Engineering, VIT UNIVERSITY. The project is the result of work carried out under the supervision of Dr. Archana Giri, Head, CBT, IST, and JNTU-HYDERABAD.

A. Srikanth

1

2

Abstract
Avian Influenza outbreaks among poultries are occur world wide from time to time with increasing Global pandemics. The strategies to prevent and test for avian influenza are becoming increasingly more important. One strain of avian influenza, H5N1, has already crossed the species border into humans. We propose a method for developing a subunit vaccine and influenza tests using Bacteria as the chassis, respectively. This system is easily amendable for targeting the two main antigens presented on the exterior of the influenza virus: Hemagglutinin and Neuraminidase. The purification and influenza vaccine & Diagnostics development. Recombinant envelop protein of H5N1, 55KDa of rNA1, was successfully produced in prokaryotic system by using pRSET/A expression vector, and the expression was optimized by IPTG, this shows the ideal condition to obtain high yield of rNA1 protein. The result of purification showed that the expressed protein purified form of rNA1.The quantification analysis through calorimetric assay revealed yield of recombinant protein rNA1 protein respectively. This

References: 1. World Health Organization: Memorandum on the classification of influenza viruses: revised system of nomenclature. Bull WHO 45: 119-124, 1971. 2. Maassab HF, Kendal AP, Davenport FM: Hybrid formation of influenza virus at 25°. Proc Soc Exp Biol Med 137: 768-773, 1972.] 3. Garcia, A., Johnson, H., Srivastava, D.K., et al., 1998. Efficacy of inactivated H5N2 influenza vaccines against lethal A/Chicken/Queretaro/19/95 infection. Avian Diseases, 42 (2), 248-256. 4. Bergmann, M., A. Garcia-Sastre, E. Carnero,H. Pehamberger, K. Wolef, P. Palese and T. Muster. 2000. Influenza virus NS1 protein counteracts PKR-mediated inhibitor of Replication. J. Virol. 74: 6203-6206. 5. Burgui, I., T. AragÛn, J. OrtÌn and A. Nieto. 2003. PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes. J. Gen. Virol. 84: 3263-3274. 6.Chien, C., Y. Xu, R. Xiao, J.M. Aramini, P.V.Sahasrabudhe, R.M. Krug and G.T. Montelione. 2004. Biophysical characterization of the complex between double- stranded RNA and the N-terminal domain of the NS1protein from influenza A virus: evidence for a novel RNA-binding mode. Biochemistry. 43:1950-1962. 7. Egorov, A., S. Brandt, S. Sereinig, J. Romanova,B. Ferko, D. Katinger, A. Grassier, G.Alexandrova, H. Katinger and T. Muster.1998. Transfect influenza A viruses with long Deletions in the NS1 protein grow efficiently in Vero cells. J. Virol. 72: 6437-6441. 66 8. Gaberc-Porekar, V. and V. Menart. 2001. Review perspectives of immobilized-metal affinity chromatography. J. Biochem. Biophys.Method. 49: 335-360. 9.García-Sastre, A., A. Egorov, D. Matassov, S.Brandt, D.E. Levy, J.E. Durbin, P. Palese and T.Muster. 1998. Influenza A virus lacking theNS1 gene replicates in interferondeficient systems. Virology 252: 324-330. 10. Hatada, E. S. Saito and R. Fukuda. 1999. Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells. J. Virol. 73: 24252433. 11. Hoffmann, E., J. Stech, Y. Guan, R.G. Webster and D.R. Perez. 2001. Universal primer set for the full-length amplification of all influenza viruses. Arch. Virol. 146: 2275-2289. 12. Israrul, H.A., K. Byungjoon, F.A. Osorio and A.K.Pattnaik. 2006. Influenza of Nlinked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigen city, and ability to induce neutralizing antibiodies, J. Virol. 80: 3994-4004. 13. Murayama, R., Y. Harada, T. Shibata, K. Kuroda,S. Hayakawa, K. Shimizu and T. Tanaka.2007. Influenza A virus non-structural protein 1 (NS1) interacts with cellular Multifunctional protein nucleoli duringinfection.Biochem. Biophysics. Res. Common. 362: 880-885. 14. Palese, P. and J.F. Young. 1982. Variation of influenza A, B and C viruses. Science 215:1468-1474. 15. Seo, S.H., E. Hoffmann and R.G. Webster. 2004.The NS1 gene of H5N1 influenza viruses circumvents the host anti-viral cytokine responses. Virus Res. 103: 107-113. Spencer, K., F.A. Osorio and J.A. Hiscox. 2007. 67 16. Recombinant viral proteins for use in diagnostic ELISAs to detect virus infection. Vaccine 25: 5653-5659. 17. Wang, W., K. Riedel, P. Lynch, C. Chien, G.T.Montelione and R.M. Krug. 1999. RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimmers structure and a small number of specific basic amino acids. RNA. 5: 195-205. Wu, R., S. Hu, Y. Xiao, Z. Li, D. Shi and D. Bi.2007. 18. Development of indirect enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection and quantification of antibodies against avian influenza virus. Vet.Res. Comm. 31: 631-641. 19. Young, J.F., U. Desselberger, P. Palese, B.Ferguson, A.R. Shatzman and M. Rosenberg.1983. Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli. Proc. Natl. Acad. Sci. USA. 80: 6105-6109 68 69