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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 222-228 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) Cloning, Expression and Purification of Haemagglutinin and Neuraminidase gene of highly Pathogenic Avian Influenza H5N1 in Escherichia coli M. Subathra 1 , P. Santhakumar 2 , P. Pardhasaradhi 1 , M. Lakshmi Narasu 1* , Chandrani Chakravarty 3 and Sunil K. Lal 3 1 Centre for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, India 2 Indian Immunologicals Ltd, Hyderabad, India 3 Virology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India *For Correspondence – mangamoori@gmail.com Abstract The looming influenza virus pandemic requires simple and quicker vaccination strategies to prevent higher mortality and morbidity both in chickens and in humans. The process of current influenza vaccines manufacturing using embryonated eggs cannot help in controlling a future pandemic as it is too slow and the yield obtained by these methods are quit lower. In this study, we have developed a bacterial expressed rHA and rNA for using as a subunit vaccine against avian influenza which could also be used as a diagnostic tools against avian influenza. The HA and NA genes were cloned individually into Escherichia coli expression vector pRSETA. The rHA and rNA were expressed in E.coli and were purified using Ni-NTA chromatography. The haemagglutinin activity of the E.coli expressed rHA was analyzed against various RBCs. The rHA and rNA expressed E.coli can be used as a quicker and cheaper subunit vaccine candidate against avian influenza and also as a tools for diagnosis. Key words: Highly pathogenic avian influenza, haemagglutinin, neuraminidase, vaccine, diagnosis. Introduction Avian influenza is a highly contagious disease caused by type A influenza virus which 222 belongs to the family Orthomyxoviridae (1). The highly pathogenic avian influenza (H5N1) virus is a significant threat not only to the poultry industry but for human too. The 1997 H5N1 outbreak in Hong Kong clearly indicated that these viruses can transmit efficiently from poultry to humans and has the potential to cause high mortality in both hosts (2). It repeatedly proven its ability to spread in humans as multiple episodes of human transmission associated with high mortality have been reported. The effective control of the disease can be achieved either by vaccination or by antiviral drugs. On the other hand, rapidly evolving drug resistance among various highly pathogenic isolates showing resistance to antiviral drugs amantadine and rimantadine (3,4) necessitates the development of potential broad spectrum prophylactic vaccines for newer strains. Currently licensed seasonal influenza vaccines is an egg based vaccine which are only partially protective, particularly in populations at highest risk of severe disease, the very young and the elderly. In addition, there is a need for novel approaches for enhancing immune responses to emerging influenza isolates. However, more research is needed in the discovery of such vaccines, adjuvants, and dosing regimens to be able to supply the world with a safe and effective vaccine against avian influenza viruses. Majority of these 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) vaccine development targets the two major structural antigens namely hemagglutinin (HA) and neuraminidase (NA). Antibodies to HA seem to play a major role in controlling the disease while NA is reported to assist in controlling virus replication but, it does not protect an animal from being infected (5,6). On the other hand, there are 16 HA subtypes and 9 NA subtypes have already been reported. The newer avian influenza virus can posses one among many probabilities of HA and NA (7). There are several alternative strategies have been developed to produce pandemic and seasonal influenza vaccines which involves mammalian cell culture and baculovirus expression systems. Several studies have demonstrated the use of HA and NA either in the form of whole virus or expressed in various platform such as baculovirus and DNA vaccine expressing HA and NA aid partial protection in animals. None the less, the development of large quantities of such vaccines in mammalian cell culture and baculovirus may not fulfill the necessity of a pandemic as millions and millions of vaccine doses would be required. Few reports demonstrated the use of yeast as an expression system for producing avian influenza antigens (8,9,10,11) which can be used as potential pandemic vaccine candidates. However, expression in yeast based system consumes considerable time for producing bulk vaccines. Hence, development of such vaccine in a bacterial expression system may be an attractive alternative to produce faster, cheaper and bulk vaccines. Development of avian influenza HA in E.coli expression system has already been described (12) yet, not many reports describe the development of such cheaper and quick vaccine against avian influenza. Furthermore, the recombinant vaccines produced in bacteria will not only avoid the use of live virus but also expected to help in reducing complications associated with the whole virus vaccines (13). In an attempt to produce a faster and safer vaccine against avian influenza, the aim of this study was to examine the feasibility of E.coli Subathra et al 223 expressed avian influenza HA and NA as a subunit vaccine against potential influenza pandemic. The HA and NA gene of H5N1 strain (A/Hatay/2004/H5N1) was cloned into E.coli expression vector, the protein was expressed and purified from E.coli. The possibility of using the rHA and rNA as a vaccine candidates discussed. Material and methods Strains, Plasmids and culture conditions : The cDNA of HA and NA gene of Avian Influenza virus H5N1 strain (A/Hatay/2004/H5N1) was obtained from the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. Escherichia coli TOP10 (F– mcrA Δ(mrr-hsdRMS-mcrBC) Δ80lacZΔM15 ΔlacX74 recA1 araD139 Ä(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG) cells were used for all the cloning and plasmid propagation work and BL21(DE3) pLysS (F– ompT hsdSB(rB–, mB–) gal dcm (DE3) pLysS (CamR) was used for the expression of rHA and rNA genes and the bacterial cells were grown and maintained in LB broth and/or LB agar containing appropriate antibiotics. Cloning of HA and NA into pRSET A vector: The HA and NA gene of avian influenza H5N1 was PCR amplified from the cDNA using specific primers employing standard PCR protocols. The gene encoding NA was amplified using forward primer (5’- ATCGGCTAGCATGA ATCC AAATCAG AAGATAATAA-3’) and a reverse primer (5’- ATCGAAGCTTCT ACTTGTCAA TGGTGAATG-3’) consisting NheI and HindIII restriction endonuclease sites respectively. The gene encoding HA was amplified using forward primer (5’- ATCGGCTAGCAT GGAGAAAATA GTGCTTCT-3’) and a reverse primer (5’- ATCGAAGCTTTTAAATGCAAATTCTGCATTG- 3’) consisting NheI and HindIII restriction endonuclease sites (underlined) respectively. A PCR reaction mixture containing 50 ng of cDNA, 1xPCR reaction buffer, 200µM of each dNTPs, 25 picomoles of each forward and reverse primers specific for HA and NA, 0.5 units of Phusion DNA polymerase enzyme was prepared
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