CLONING AND EXPRESSION OF Lb-PROTEASE FROM cDNA CLONE OF FOOT-AND- MOUTH DISEASE VIRUS

Swaroop Sarkar; V.V.S. Suryanarayana; S.R.Madhan Shankar

doi:10.29121/granthaalayah.v5.i9(SE).2017.2258

Authors

Swaroop Sarkar Research and Development Centre, Bharathiar University, Coimbatore- 641046, India & Indian Veterinary Research Institute, Hebbal, Bangalore – 560024, India
V.V.S.Suryanarayana Indian Veterinary Research Institute, Hebbal, Bangalore – 560024, India
S.R.Madhan Shankar PSG College of arts and science, Coimbatore, Tamil Nadu- 641046, India

DOI:

https://doi.org/10.29121/granthaalayah.v5.i9(SE).2017.2258

Keywords:

Cloning, Expression, FMDV Virulence Factor, Lb-Protease

Abstract [English]

Foot-and –Mouth disease virus (FMDV) is a positive sense RNA virus and the genome codes for single polyprotein. The FMDV L protein is located at the N terminus of the polyprotein and is the first gene product released from the nascent polyprotein. The leader L protease which is involved in pathogenesis has two known functions: (i) auto-catalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. To explore the role of L protease in FMDV pathogenesis we generated synthetic FMDV genome lacking the L gene. The gene was amplified from an infectious cDNA clone of serotype Asia1. Primers corresponding to L protease were designed based on the sequence available in the data base. An amplified DNA of 546bp was purified and cloned into pET28 cloning vector. The sequence analysis revealed the presence of single Open Reading Frame (ORF) encoding a protein of 173 amino acid residues. The sequence alignment using BLAST search in NCBI gene Bank showed 91% homology with FMDV strain A isolate IND17/77 L protease gene. The recombinant plasmids pETLb was transferred into BL21 (DE3) pLysS cells and the IPTG induced expressed protein of 25 KDa was purified by nickel affinity column as per the manufacturer’s protocol (Sigma, USA). The specificity of the expressed protein in was confirmed by western blotting using convalescent cattle serum/ rabbit anti-bovine horse radish peroxidase conjugate and O-Dianisidine Dihydrochloride substrate.

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References

Francisco S,Margarita S,Miguel A J,etal.Foot-and-Mouth disease virus:along known virus, but a current threat [J]. Vet res, 2001,32:12-30 DOI: https://doi.org/10.1051/vetres:2001106

Knlowles, N.J. and Samuel, A.R. (2003) molecular epidemiology of foot and mouth disease virus. Virus res, 91(1):65-80 DOI: https://doi.org/10.1016/S0168-1702(02)00260-5

Ko, Y.J., Jeoung, H.Y., Lee, H.S., Chang, B.S., Hong, S.M., Heo, E.J., Lee, K.N., Joo, H.D., Kim, S.M., Park,J.H. and Kweon,C.H.(2009) A recombinant protein-based ELISA for detecting antibodies to foot-and-mouth disease virus serotype Asia1. J.Virol.Methods, 159(1):112-118. DOI: https://doi.org/10.1016/j.jviromet.2009.03.011

Rodriguez LL, Grubman MJ. Foot and mouth disease virus vaccines. Vaccine. 2009;4(Supplement 4):D90–4. DOI: https://doi.org/10.1016/j.vaccine.2009.08.039

Gibbs P. The foot-and-mouth disease epidemic of 2001 in the UK: implications for the USA and the “war on terror”. J Vet Med Educ. 2003;30:121–32. DOI: https://doi.org/10.3138/jvme.30.2.121

Sumption K, Rweyemamu M, Wint W. Incidence and distribution of footand-mouth disease in Asia, Africa and South America; combining expert opinion, official disease information and livestock populations to assist risk assessment. Transbound Emerg Dis. 2008;55:5–13. DOI: https://doi.org/10.1111/j.1865-1682.2007.01017.x

Anonymous. Foot-and-mouth disease. In: Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees). Paris, France: World Organization for Animal Health (OIE); 2008.

Rodriguez LL, Gay CG. Development of vaccines toward the global control and eradication of foot-and-mouth disease. Expert Rev Vaccines. 2011;10:377–87 DOI: https://doi.org/10.1586/erv.11.4

T.sarvanan, G.R.Reddy, H.J.Dechamma, V.V.S.Suryanarayana.(2003) Construction of gnome-length cDNA for foot-and-mouth disease virus serotype Asia 1 IND 63/72 vaccine strain.IJBMBR,2(2).

Bradford M M, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem, 72 (1976) 248-254.

Laemmli U K, Cleavage of structural protein during the analysis of head of bacteriophage T4, Nature (Lond), 227 (1970) 680-685.

Brown, C. C., M. E., Piccone, P.W.Mason, T.S.C. Mckenna, and M.J.Grubman. 1996. Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle. J.Virol. 70:5638-5641. DOI: https://doi.org/10.1128/JVI.70.8.5638-5641.1996