Purification of Citrus Tristeza Virus and Generation of Monospecific Polyclonal Antiserum
Symptomatology, molecular weight of coat protein (CP) determination and purification procedure for citrus tristeza virus (CTV) antigen were studied to generate monospecific polyclonal antiserum. Results of survey indicated that CTV was found to attack citrus varieties such as C. grandis, C. aura...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2002
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/10621/1/FP_2002_32.pdf http://psasir.upm.edu.my/id/eprint/10621/ |
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| Summary: | Symptomatology, molecular weight of coat protein (CP) determination and
purification procedure for citrus tristeza virus (CTV) antigen were studied to
generate monospecific polyclonal antiserum. Results of survey indicated
that CTV was found to attack citrus varieties such as C. grandis, C.
aurantifolia, C. reticulata, C. hystrix, C. nobilis and C. sinensis with variable
symptoms and degrees of severity. Virus complex varied among citrus
varieties and cultivars, and there were four important sub isolates found,
namely mandarin stem pitting, orange stem pitting, pomelo small fruit, and
pomelo mild isolate.
In partial purification step, antigen was concentrated by polyethylene glycol
(PEG) precipitation followed by low speed centrifugation. Semi purified antigen was then finally purified by sodium dodecyl sulphate polyacrylamide
gel electrophoresis (SOS-PAGE). A major protei n band containing CP was
excised and eluted using elution buffer containing 0.25 M Tris-HCI pH 6.8+0.1% SOS. With a starting material consisting of 50-gram bark tissue of
semi dormant flush, 750 ug of eluted CP as monospecific antigen was
obtained. The use of PEG and NaCI for virus precipitation combined with low
speed centrifugation in semi purification step, then followed by
electrophoresed of semi purified virus preparation in final purification step
effectively minimized virus losses during purification processes and
minimized possible contamination of plant components in the final
immunogen.
Monospecific polyclonal antiserum against citrus tristeza virus was
generated using the viral CPo Upon SOS-PAGE of local CTV isolate
designated as UPM/T002, two protein bands specific for CTV with
molecular weight of 25 kOa and 33 kOa were obtained. The major band
with molecular weight of 25 kOa that reacted strongly with commercial
polyclonal antibody in double antibody sandwich enzyme-linked
immunosorbent assay (OAS-E LlSA), was used as the antigen for injection
into female White Leghorn chicken. |
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