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Asian Journal of Agricultural Research
  Year: 2014 | Volume: 8 | Issue: 2 | Page No.: 114-121
DOI: 10.3923/ajar.2014.114.121
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Expression of Recombinant Coat Protein (CP) of Citrus tristeza virus for Polyclonal Antibodies Production

Surafel Shibru, Ampaiwan Paradornuwat and Srimek Chowpongpang

Citrus tristerza virus (CTV) is one of the most destructive diseases in citrus growing areas of Thailand. CTV coat protein gene (CTV-CP) of Thailand MK-50 isolate which is cloned in pQE30 vector, cloned into pET160/GW/D-TOPO vector (INVITROGEN) allowing fusion of the protein with N-terminal 6XHis tag for easier protein purification and transformed into BL21 starTM (DE3) expression E. coli strain. The expression was induced by 1 mM Isopropyl-beta-D-thiogalactopyranoside (IPTG), followed by further growth for 4 h. A rabbit was immunized intradermally with a mix of recombinant CTV Coat Protein (CTV-CP) and complete Freund’s adjuvant as primary immunization and the remaining six immunization was done with mix of incomplete Freund's adjuvants within two weeks interval. The animal was bled prior to each immunization and the titer level was examined for binding recombinant CTV-CP by the indirect ELISA (Enzyme-Linked Immunosorbent assay). The collected serum was used to determine the titer as primary antibody. The result indicated that the titer level was gradually increase and reach to pick after third to sixth immunizations then lowered. The collected sera can able to detect an antigen of 20 ng at 31, 250 fold dilution and the purified polyclonal antibodies also can able to detect positive plant samples.
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How to cite this article:

Surafel Shibru, Ampaiwan Paradornuwat and Srimek Chowpongpang, 2014. Expression of Recombinant Coat Protein (CP) of Citrus tristeza virus for Polyclonal Antibodies Production. Asian Journal of Agricultural Research, 8: 114-121.

DOI: 10.3923/ajar.2014.114.121






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