Expression and purification of coat protein of citrus tristeza virus
Citrus tristeza virus (CTV) polyclonal antibodies produced either from the recombinant coat protein (CP) of CTV or extracted virus from midrib used for the detection of virus. Compared with intact virion procedure, the use of CP antigen resulted in highly specific polyclonal antibodies. CTV coat protein gene (CTV-cp) cloned in pQE30 vector and transformed to DH5α containing 666bp long from Thailand MK-50 isolate was amplified with a forward primer CTV-CP1 (5’ CAC CGA CGA AAC AAA GAA ATT GAA GAA CA 3’) and a reverse primer CTVCP2 (5’ TCA ACG TGT GTT AAA TTT CCC AAG C 3’) and cloned into TOPO vector and transformed to TOP10 E. coli competent cell. Six colonies of TOP10 E. coli were selected and checked for the appropriate insertion of cp gene with PCR using T7F (5’ TAA TAC GAC TCA CTA TAG GG 3’) as forward primer and CTVCP2 as reverse primer. Two colonies having appropriate 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. One of the colonies was selected and used for mass production of recombinant protein and the produced protein was purified using Ni-NTA resin. The result indicated that the expression of recombinant CP was obtained only for cloned CTV-cp gene in TOPO vector within BLD21 star (DE3) E. coli cell and inducing protein for 4hours after addition of 1mM IPTG were given optimum amount of recombinant protein expression. The recombinant CTV-CP was highly bound to Ni-NTA resin and only eluted when washed with low pH buffer during the purification, and can be used for polyclonal antibodies production.
Keywords: CTV, cp gene, SDS-PAGE