<?xml version="1.0" encoding="ISO-8859-1"?><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance">
<front>
<journal-meta>
<journal-id>0185-3309</journal-id>
<journal-title><![CDATA[Revista mexicana de fitopatología]]></journal-title>
<abbrev-journal-title><![CDATA[Rev. mex. fitopatol]]></abbrev-journal-title>
<issn>0185-3309</issn>
<publisher>
<publisher-name><![CDATA[Sociedad Mexicana de Fitopatología A.C.]]></publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id>S0185-33092025000200001</article-id>
<article-id pub-id-type="doi">10.18781/r.mex.fit.2405-00</article-id>
<title-group>
<article-title xml:lang="es"><![CDATA[Clonación, expresión y detección serológica de la putativa ARN polimerasa del Virus de la meleira de la papaya variante mexicana en Escherichia coli]]></article-title>
<article-title xml:lang="en"><![CDATA[Cloning, expression, and serological detection of the putative RNA polymerase of Papaya meleira virus Mexican variant in Escherichia coli]]></article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Serra]]></surname>
<given-names><![CDATA[Wendy]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Arguelles-Quintal]]></surname>
<given-names><![CDATA[Jeanin A.]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Carrillo-Pech]]></surname>
<given-names><![CDATA[Mildred]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Toriz]]></surname>
<given-names><![CDATA[Alethia F.]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Castaño]]></surname>
<given-names><![CDATA[Enrique]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Islas-Flores]]></surname>
<given-names><![CDATA[Ignacio]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Lopez-Ochoa]]></surname>
<given-names><![CDATA[Luisa A.]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
</contrib-group>
<aff id="Af1">
<institution><![CDATA[,Centro de Investigación Científica de Yucatán, A.C.  ]]></institution>
<addr-line><![CDATA[Mérida Yucatán]]></addr-line>
<country>Mexico</country>
</aff>
<aff id="Af2">
<institution><![CDATA[,Centro de Investigación Científica de Yucatán, A.C.  ]]></institution>
<addr-line><![CDATA[Mérida Yucatán]]></addr-line>
<country>Mexico</country>
</aff>
<pub-date pub-type="pub">
<day>00</day>
<month>00</month>
<year>2025</year>
</pub-date>
<pub-date pub-type="epub">
<day>00</day>
<month>00</month>
<year>2025</year>
</pub-date>
<volume>43</volume>
<numero>2</numero>
<copyright-statement/>
<copyright-year/>
<self-uri xlink:href="http://www.scielo.org.mx/scielo.php?script=sci_arttext&amp;pid=S0185-33092025000200001&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.org.mx/scielo.php?script=sci_abstract&amp;pid=S0185-33092025000200001&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.org.mx/scielo.php?script=sci_pdf&amp;pid=S0185-33092025000200001&amp;lng=en&amp;nrm=iso"></self-uri><abstract abstract-type="short" xml:lang="es"><p><![CDATA[RESUMEN   Antecedentes/Objetivo. La enfermedad de la meleira en papaya en México está asociada al Virus de la meleira de la papaya variante mexicana (PMeV-Mx) y se caracteriza por la exudación espontánea de látex en los frutos. El ORF2 de PMeV-Mx codifica una proteína que tiene motivos de ARN polimerasa dependiente de ARN (RdRp) la cual es esencial para la replicación viral. El objetivo de este estudio fue desarrollar un método para la producción y purificación de la proteína recombinante codificada por el ORF2 de PMeV-Mx (pORF2) en Escherichia coli y generar anticuerpos específicos para su detección.   Materiales y Métodos. El genoma de PMeV-Mx se analizó utilizando UGENE para predecir los ORFs e identificar el posible sitio deslizante. Se utilizaron plantas de papaya inoculadas con látex de frutos de plantas sintomáticas como reservorio del virus, la infección por PMeV-Mx se confirmó mediante RT-PCR. El ORF1 y ORF2 se amplificaron por PCR a partir de ADNc de papayas infectadas. Ambos se clonaron en el vector pGEM-T Easy y se transfirieron a pDONR221 y pDEST17 para su expresión en E. coli. La proteína recombinante 6xHis-pORF2 se expresó en la cepa BL21 de E. coli y se purificó bajo condiciones desnaturalizantes. La proteína purificada se utilizó para generar anticuerpos policlonales. La especificidad del antisuero y sus diluciones óptimas de trabajo se evaluaron mediante ensayos de inmunodetección, utilizando la proteína recombinante como diana y proteínas etiquetadas con His como controles negativos.   Resultados. Se logró la expresión y purificación de la proteína recombinante 6xHis-pORF2 de PMeV-Mx. Se detectó una banda de aproximadamente ~46,5 kDa, consistente con su peso molecular estimado. La expresión de la proteína se observó entre 2 y 6 horas después de la inducción. El análisis por western blot confirmó la presencia de la etiqueta de Histidinas y la integridad de la proteína recombinante. La purificación con resina Ni-NTA resultó en una banda intensa de ~46,5 kDa, junto con bandas menores de 15 a 150 kDa. Se generaron anticuerpos policlonales contra pORF2, los cuales reconocieron específicamente la proteína purificada en los ensayos de inmunodetección, con detección en diluciones de 1:500 a 1:3000. No se observó reactividad cruzada con los controles negativos, aunque se detectó una banda inespecífica de ~75 kDa en extractos de E. coli.   Conclusión . Se estableció un protocolo para la expresión, detección y purificación de la proteína recombinante 6xHis-pORF2 de PMeV-Mx en E. coli. Los anticuerpos policlonales generados en conejo reconocieron la proteína de interés en extractos proteicos de células bacterianas, aunque se requiere una mayor optimización para mejorar la especificidad. Estos anticuerpos contribuirán al desarrollo futuro de herramientas de diagnóstico y a la caracterización de la proteína en plantas.]]></p></abstract>
<abstract abstract-type="short" xml:lang="en"><p><![CDATA[ABSTRACT   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.]]></p></abstract>
<kwd-group>
<kwd lng="es"><![CDATA[Meleira de la papaya]]></kwd>
<kwd lng="es"><![CDATA[proteína recombinante]]></kwd>
<kwd lng="es"><![CDATA[anticuerpos]]></kwd>
<kwd lng="es"><![CDATA[virus similares a umbravirus]]></kwd>
<kwd lng="es"><![CDATA[PMeV-Mx.]]></kwd>
<kwd lng="en"><![CDATA[Papaya meleira]]></kwd>
<kwd lng="en"><![CDATA[Recombinant proteins]]></kwd>
<kwd lng="en"><![CDATA[Antibodies]]></kwd>
<kwd lng="en"><![CDATA[Umbra-like viruses]]></kwd>
<kwd lng="en"><![CDATA[PMeV-Mx]]></kwd>
</kwd-group>
</article-meta>
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