Expression of recombinant coat protein (CP) of Citrus tristeza virus

Citrus tristeza virus (CTV) is one of the major threats for the production of citrus worldwide. Polyclonal antibodies either produced from the recombinant coat protein (CP) of CTV or purified viral particles from infected midrib used for the detection of virus. In comparison, the purified virion was a laborious procedure and with contamination from plant proteins, when use of recombinant CP antigen resulted in highly specific polyclonal antibodies without cross reaction with plant protein. A CTV cp gene clone contained 666bp long from Thailand Msh-141 isolate collection was used for expression of recombinant protein. A forward primer CTV-CP1 and reverse primer CTV-CP2 was designed to amplify and clone and express the cp gene into PET160/GW/D-TOPO vector and transformed to alphaDH5 E. coli competent cell. Two clones harboring the correct orientation insertion were selected for transformation into BLD21 star (DE3) expression E. coli cell and their recombinant protein expressions capacity and optimum length of time were studied after inducing with 1mM IPTG. The optimum time for recombinant protein production was investigated. The large scale production of recombinant CP and produced protein was purified using Ni-NTA resin. Result on sequence analysis of extracted plasmid used for mass protein was similar to MSh-141 isolate. Moreover, the expression of recombinant CP was very high for the cloned CP with pET160/GW/D-TOPO vector expressed in BLD21 star (DE3) E. coli cell compared with the initial clone induced. Inducing protein for 4 hours after addition of 1m MIPTG gave optimum amount of recombinant protein expression with molecular weight approximately 25 kDa which is similar to previous research works. The purified protein from this experiment will be used to immunize rabbit or chicken for production of polyclonal antibodies for detection of CTV using ELISA or immunochromatography.