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e-Proceedingof1stInternationalNastaranCancerSymposium-2015 www.nastaransymposium.com/proceeding2015 ConstructionofRecombinantTransgenicBacteria ExpressingHumanCollagenase3(MMP13)Gene SamanehTalebi1 ,MehranGholamin 2 1. DepartmentBiology,DamghanBranch,IslamicAzadUniversity,Damghan,Iran 2. DivisionofHumanGeneticsLab,ImmunologyResearchCenterAvicennaResearchInstitute,MashhadUniversityofMedical Sciences,Mashhad,Iran. Abstract Esophageal carcinoma is one of the most common malignancies with high cancer-related morbidityandmortalityworldwide.Despiteadvancesinsurgicaltechniquesandtreatment, the prognosis of esophageal cancer remains poor, with very few long-term survivors. Therefore, more specific and sensitive biomarkers for diagnosis and targeted therapy of ESCCareurgentlyneeded.Collagenase3isanenzymethatinhumansisencodedbythe MMP13gene.Expressionof,matrixmetalloproteinase(MMP)-13,hasbeenimplicatedinthe progressionofseveraltypesofhumancancer.(MMP13)isahighlyregulatedzinc-dependent endopeptidase and has been reported to be associated with vascular invasion and lymph node metastasis and predicts poor outcome for relatively early stage in esophageal squamous cell carcinoma (ESCC) patients. That’s the elevated level of preoperative MMP-13, found to be associated with tumor progression and poor survival in patients with ESCC.Accordingtotheimportanceofthisproteininesophagealsquamouscellcarcinoma (ESCC)patients,producingofrecombinantMMP-13inourcountryisveryessential.cDNA was synthesized by RT-PCR from total RNA. The target sequence was amplified by PCR using designed primers which had restriction sites.Analysis of PCR product was done by electrophoresis on agarose gel. Amplified sequence was ligated to pET21-b(+) vector. RecombinantplasmidwastransformedtoE.coliTop10.Clonedplasmidsweresequenced. Recombinant plasmid was transformed to BL21 for protein expression. The expression of protein was induced by IPTG. The expressed protein was analyzed by SDS-PAGE. To demonstrate the nature of the protein, western blot was performed. Recombinant MMP13 protein with a molecular weight of 14 kDa was expressed by pET-21b (+) bacteria BL21 (DE3) expression system. The bacteria can produce MMP13 protein in large number by properinducer.Westernblotwasperformedtoverifythetypeofprotein.ProducedMMP13 proteincanbeusedforresearchesanddiagnosticpurposes.Sowecanusethisrecombinant proteintoproducingesophaguscarcinoma’sdiagnostickitbasedonELIZAtechnique. Alsowecanstrengthentheimmunesystemtopreventcancerbyusetherecombinantprotein asavaccine. Keywords:RecombinantProtein,MMP13,TOP10,pET-21b(+),BL21(DE3) CorrespondingAuthor:MehranGholamin Email:[email protected]
