Microbiologia Médica e Diagnóstico

In Silico Analysis, Cloning and Expression of Recombinant CD166 in E. coli BL21 (DE3) as a Marker for Detection and Treatment of Colorectal Cancer

Abstract

Vahid Marmari, Habibollah Mahmoodzadeh, Hassan Dana, Ghanbar Mahmoodi Chalbatani, Ali Mazraeh, Ali Ghamari, Fateme Moazzen, Mohammad Ebrahimi and Narges Mehmandoost

Introduction: Colorectal cancer is the third most common type of tumors, with more than 1.2 million new cases resulted in 600 thousand deaths annually and ranks fourth in terms of mortality worldwide. The activated leukocyte cell adhesion molecule (ALCAM) also called CD166 is a marker of colorectal cancer (CRC) stem cells. The expression of CD166 increase in colorectal cancer. Also with advancement of illness in different stages of cancer, this expression increased. So, it could be a reasonable marker for Detection and Treatment of Colorectal cancer. The purpose of this study is to produce recombinant protein CD166 for cancer therapy or early detection of colorectal cancer cells.

Methods: In this study, the sequence of CD166 was optimized for expression in E. coli using online tools and cloned into pET28a as an expression vector. The recombinant pET28a was transformed into the E. coli BL21DE3 using heat shock method and expression of recombinant CD166 was examined using SDS-PAGE.

Results: The synthetic gene of CD166 was located between NcoI/BamHI and XhoI restriction sites and cloned into pBSK (+) vector. The presence of this gene in pET28a was determined by colony and confirmed by restriction digestion. Gene of CD166 were expressed in E. coli BL21 DE3. The results of the SDS-PAGE technique confirmed the expression of recombinant 53 kDa CD166 in a bacterial expression system.

Conclusion: A portion of the CD166 gene was expressed as a recombinant in E. coli. This could be a good candidate to produce a vaccine for cancer therapy or colorectal cancer diagnostic test.