PRODUCTION OF POLYCLONAL ANTIBODIES SPECIFIC TO CITRUS TRISTEZA VIRUS

Citrus tristeza virus (CTV) is one of the most destructive and economically important diseases of commercial citrus worldwide. In this study, the coat protein (cp) gene of CTV was subcloned into PQE-30 vector at Bam HI and Hind-III restriction sites. The constructed plasmid that called pRMAF2 was transformed into Escherichia coli M15 strain for its expression and production of fusion protein for CTV-cp gene. The suitable condition for inductions of CTV-cp was in the presence of 1 mM IPTG and overnight incubation time. The bacterial culture was harvested and the expressed fusion protein(s) was purified by QIA express kit. Polyclonal antibodies (PAbs) were raised by injection of the purified fusion protein(s) into two white mice followed by western blotting analysis using the expressed fusion protein(s) and the CTV-infected citrus extracts as controls. It can be concluded that the immuno-enzymatic detection based on the produced antibodies against CTV-cpORF fusion proteins were safe, speed and sensitive for the detection of the virus. INTRODUCTION Citrus is one of the most widely grown and economically important fruit crops in the world for generation of income, foreign trade and nutrition. The world citrus production (about 100 million metric tons) has been increasing steadily to accommodate growing demands of domestic and international markets (Çevik, 2001). CTV (Citrus triteza virus) is a member of the Closteroviridae, it is the causal agent of the most economically important viral disease of citrus worldwide (Bar-Joseph et al., 1989). The virus causes one of the most destructive diseases of citrus (Bar-Joseph et al., 1989). CTV isolates differ in genome sequences (Vives et al., 1999) and biological properties, such as symptoms induced on different hosts and aphid transmissibility. Some isolates caused decline of citrus varieties grafted on sour orange rootstock (C.aurantium L.), while other causes are stem pitting, lack of vigor in stem as well roots and reduce yield (BarJoseph et al., 1989) while few isolates induce very mild symptoms or symptomless, even in the most sensitive citrus species. CTV virion is flexuous particle, about 2000 X 11 nm in size and contains a single positive-stranded genomic RNA of 19200– 19300 nucleotides with 12 ORFs that encode up to 19 polypeptides (Karasev et al., 1995; Mawassi et al., 1996). The ORFs 25 and 27 code the capsid proteins of 25 and 27 KDa, respectively (Sekiya et al., 1991). The 25 KDa CP encapsidates 95% Salama et. al., Pak. J. Biotechnol 2 of the particle length whiles the 27 KDa protein encapsi-dates one end of the particle forming a ‘rattlesnake’ structure (Febres et al., 1996). Therefore, this study was aimed to produce of expressed fusion protein against cp gene and use it as antigen in producing of specific polyclonal antibody (PAb) for virus detection in immuno-serological reactions. MATERIAL AND METHODS 1. Subcloning of CTV-ORF-cp gene and bacterial transformation: The pRMAF1 (pGEM-T easy vector + cp-ORF) was separately digested with Bam H1 and Hind III restriction endonuclease enzy-mes and the released fragment was then subcloned into digested PQE-30 vector (QIAexpress® Type IV kit), with the same previous enzymes. Recombinant plasmid (pRMAF2PQE-30 + cp-ORF) was transformed into E. coli strain M15 (pREP4) for expression and production of fusion protein as (Nour El-Din et al., (2005). 2. Purification of the 6X His-tagged cpfusion protein: After confirmation and expression processes, purification process was carried out in a large scale using nickel-nitrilotriacetic acid (Ni-NTA) batch chromatography under denaturing conditions as recommended by Rajamohan et al. (1999) and finally analyzed by SDSPAGE (Sambrook et al., 1989). 3. Determination of the specificity of purified expressed protein: Serial dilutions from the purified expressed protein were prepared and used for DAS-ELISA (Clark and Adams, 1977) using PAbs that purchased from Valanzano, Italy. 4. Production of antiserum specific to the 6X His-tagged CTV-cp fusion protein: Antiserum was produced by injecting two white mice with 100 μg\injection purified 6X His-tagged fusion protein(s) in which the first injection emulsified with an equal volume of complete freund ' s adjuvant and the second injection emulsified with an incomplete adjuvent for four subsequent intramuscular injections at weekly intervals. The blood was collected then incubated at 37 o C for 1 h and centrifuged at 4000 rpm at room temperature (RT). The antiserum was collected and stored at 4 o C until use. 5. Western blotting analysis: Western blotting analysis (Towbin et al. 1979) was carried out to determine the specificity of the produced immunoglobulin G (IgGs) to the cp-fusion protein(s) in the presence of healthy and CTV-infected citrus plants as well as the purified fusion proteins (as noninfectious positive control). After electrophoresing at 12% SDS-PAGE, the polypeptides were blotted onto a membrane (Immobilon ® PVDF membrane, Millipore cooperation, Bed ford, MA 01730) using a trans-blot apparatus (BioRad).