Construction of Recombinant Transgenic Bacteria Expressing Human Collagenase3 (MMP13) Gene

Esophageal carcinoma is one of the most common malignancies with high cancer-relatedmorbidity and mortality worldwide. Despite advances in surgical techniques and treatment,the prognosis of esophageal cancer remains poor, with very few long-term survivors.Therefore, more specific and sensitive biomarkers for diagnosis and targeted therapy ofESCC are urgently needed. Collagenase 3 is an enzyme that in humans is encoded by theMMP13 gene. Expression of , matrix metalloproteinase(MMP)-13, has been implicated in theprogression of several types of human cancer. (MMP13) is a highly regulated zinc-dependentendopeptidase and has been reported to be associated with vascular invasion and lymphnode metastasis and predicts poor outcome for relatively early stage in esophagealsquamous 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.According to the importance of this protein in esophageal squamous cell carcinoma (ESCC)patients, producing of recombinant MMP-13 in our country is very essential. cDNA wassynthesized by RT-PCR from total RNA. The target sequence was amplified by PCR usingdesigned primers which had restriction sites. Analysis of PCR product was done byelectrophoresis on agarose gel. Amplified sequence was ligated to pET21-b(+) vector.Recombinant plasmid was transformed to E. coli Top10. Cloned plasmids were sequenced.Recombinant plasmid was transformed to BL21 for protein expression. The expression ofprotein was induced by IPTG. The expressed protein was analyzed by SDS-PAGE. Todemonstrate the nature of the protein, western blot was performed. Recombinant MMP13protein 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 byproper inducer. Western blot was performed to verify the type of protein. Produced MMP13protein can be used for researches and diagnostic purposes. So we can use this recombinantprotein to producing esophagus carcinoma’s diagnostic kit based on ELIZA technique.Also we can strengthen the immune system to prevent cancer by use the recombinant proteinas a vaccine.

Keywords:

Recombinant Protein , MMP13 , TOP10 , pET-21b(+) , BL21(DE3)