Background/Objective.

Papaya meleira disease in Mexico is associated to Papaya meleira virus Mexican variant (PMeV-Mx) and is characterized by the spontaneous exudation of latex in fruits. PMeV-Mx ORF2 encodes a protein with RNA-dependent RNA polymerase (RdRp) motifs, which is essential for viral replication. This study aimed to develop a method for producing and purifying recombinant PMeV-Mx ORF2 encoded protein (pORF2) in E. coli and generate specific antibodies for its detection.

Materials and Methods.

The PMeV-Mx genome was analyzed using UGENE to predict ORFs and identify the putative slippery site. ORF1 and ORF2 were amplified by PCR from cDNA obtained from infected papaya latex, cloned into pGEM-T Easy, and subsequently transferred into pDONR221 and pDEST17 for expression in E. coli. Recombinant 6xHis-pORF2 was expressed in E. coli BL21 strain, induced with IPTG, and purified under denaturing conditions. The purified protein was used to generate polyclonal antibodies, with immunizations conducted at different time points. Antisera specificity and optimal working dilutions were evaluated by immunodetection assays, using recombinant 6xHis-pORF2 as the target and His-tagged proteins as negative controls. Additionally, papaya plants were inoculated with latex from symptomatic fruits as virus reservoir and PMeV-Mx infection was confirmed by RT-PCR.

Results.

The recombinant 6xHis-pORF2 protein of PMeV-Mx was expressed in E. coli and purified. A ~46.5 kDa band was detected, consistent with its estimated molecular weight. The protein expression increased between 2- and 6-hours post-induction with IPTG. Western blot analysis confirmed the presence of the His- tag and the integrity of the recombinant protein. Purification using Ni-NTA resin resulted in a strong ~46.5 kDa band along with light bands ranging from 15 to 150 kDa. Polyclonal antibodies against pORF2 were generated and specifically recognized the purified protein in immunoassays, with detection observed at dilutions from 1:500 to 1:3000. No cross-reactivity was observed with negative controls, but a non-specific

~75 kDa band was detected in E. coli extracts.

Conclusion

. A protocol for expressing, detecting, and purifying the recombinant 6xHis-pORF2 protein from PMeV-Mx in E. coli was established. Rabbit polyclonal antibodies recognized the target protein in bacterial fractions, though further optimization is needed to enhance specificity. These antibodies will support future diagnostic development and protein characterization in plants.

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