Purification and antiserum preparation of Celery mosaic potyvirus, and study its molecular properties
ANBAR JOURNAL OF AGRICULTURAL SCIENCES,
2010, Volume 8, Issue 4, Pages 446-452
AbstractCeMV was propagated in Datura, and purified by grinding the infected leaves in phosphate buffer 0.05 M containing 0.01 M EDTA, 0.05% DIECA, pH 8. The extract was clarified by mixture of chloroform-butanol (1:1) 25%. The virus was precipitated by PEG 20000 at 4% with 0.3 M NaCl and collected by centrifugation at 10000 rpm. for 1 hr. The absorption ratio at 260/280 nm for the purified virus was found to be 1.22 which indicate rhod shaped particles. 2.77 mg/100g leaves of purified virus was obtained. Antiserum to CeMV was obtained by 4 injections each of 0.5 mg/ml purified virus. The first three injections were intramuscularly emulsified with an equal volume of incomplete fruend’s adjuvant at interval of 7 days. The fourth booster injection intravenously 2 weeks of the 3rd one. Blood was collected through ear marginal vein one week after the last injection with titer of 1/1024. An agglutination was appeared between CeMV antiserum with samples of purified virus and extract from CeMV-infected Datura. Band of 33.5 kd was visualized on 10% polyacrylamide gel when a sample of purified virus analyzed by electrophoresis. This band was absent in extract from healthy plants submitted to the same steps of virus purification.
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