Thesis production of recombinanat coat protein and its polyclonal antibodies for Citrus Tristeza virus diagnosis.

Citrus tristeza virus (CTV). a member of the family Closteroviridae. is one of the most destructive diseases of citrus in the wouls. causing a diversity of symptoms on various scion and rootstock in combinations.since October 1998.CTV is recognized as one of a destructive disease of citrus in Thailand.There are different disease diagnosis methods used to differentiate infected plants from non-infected one.among these polyclonal antibodies were considered as economical detection tools which can be widely applied in citrus growing areas for diagnosis and management of the disease recently.polyclonal antibodies are produced from purified recombinant coat protein (CP) of CTV as an antigen Since recombinant CP antigen simulated highly spcific polyclonal antibodies without cross reaction with plant protein. A CTV coat protein (cp) gene contained 666 bp length from Thailand MK-50 isolate was cloned into PET160/GW/D-TOPO vector (Invitrogen) which possesses 6XHis tag and subsequently transformed into BL21 star (DE3) E colistrain.The optimum protein expression time for recombinanat protein was 4 hour after addition of 1mM IPTG. Large scale produced recombinanat coat protein was purified using Ni-NTA agarose resin.The concentraion of purified recombinant CTVB-CP was 1.0mg per ml and a total of 4.3mg of purified recombinant CTV-CP was purified from 1 litter of culture.Five hundred mierogram of purified recombinant CTV-CP was mixed with equal volume of complete Freund's adjuvant and subscutaneously injected to rabbit and intramuscularly to chichen as primary immunization.At two weeds interval six booster immunizations were also done by mixing 500µg of purified recombinant protein with equal volume of incomplete Freund's adjuvant.The titer level from rabbit was increased gradually and reach maximum on eighth weeds after primary immunization and remined at this level throughout the experimental period and the cause for such low respond for immunized animal could come due to the immunzation protocol used in this study. The purified immunoglobulin from rabbit was able to detect 20 and 4ng purified recombinant coat protein at 0.08 and 0.4µg/ml immunoglobulin protein concentration respectively and capable of use for diagnosis plant sample for CTV infection at 10µg/ml immunoglobulin protein concentration.