Cloning, expression and purification of recombinant RTSV and RTBV coat proteins for polyclonal antibody production

A.B. Norliza, H.Y. Lau, H. Habibuddin and C.S. Tan

Abstract

Tungro is one of the most damaging and serious diseases affecting rice production in many countries especially in the Southeast Asia including Malaysia. The disease is caused by a combination of rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). The low concentration of viruses in the infected plants makes it difficult to obtain sufficient amount of the pathogen to be used as antigen in the production of its antibodies. The genes encoding the coat proteins of the RTSV and RTBV were amplified from total DNA and converted mRNAs extracted from plants infected by both RTSV and RTBV. The amplified PCR fragments of the genes ranging from 700 to 1000 bp in sizes were purified, restriction enzyme digested and ligated into a bacterial expression plasmid vector, pRSETA. The expression of recombinant RTSV and RTBV coat protein genes via isopropyl thiogalactoside (IPTG) induction were carried out after the clones verification using restriction enzyme and DNA sequencing. The sizes of the expressed proteins were 25 – 33 kDa as estimated by migration in 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Inclusion bodies of recombinant RTSV coat proteins were purified by repeated washes with Triton while the recombinant RTBV coat proteins were purified by nickel-chelating resin under denaturing conditions. Purified inclusion bodies of RTSV and RTBV coat proteins were solubilised and used to generate polyclonal antibody. These antibodies could specifically be used in ELISA and Western blotting analysis for detection of both viruses.

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