Resistance of Cloned ۱F۵ Chimeric Anti-CD۲۰ Antibody Heavy-Chain Gene to DNA Polymerase due to a Predicted Hairpin Structure

Background: Formation of secondary structure such as DNA hairpins or loops may influence molecular genetics methods and PCR based approaches necessary for genetic engineering, in addition to gene regulation. Materials and Methods: A polymerase chain reaction with splice overlap extension (SOE-PCR) was used to create fully synthetic ۱F۵ chimeric anti-CD۲۰ heavy- and light-chain genes. The chimeric genes were cloned into the pCR-Blunt II-TOPO vector following by cloning into the pBudCE۴.۱ expression vector. Prediction of secondary structure was performed with the Vienna RNAfold webserver. PCR and sequencing across the predicted secondary structure of chimeric ۱F۵ heavy-chain gene was performed with multiple protocols for standard and GC-rich templates. Results: In our attempt to design vectors aimed to generate mouse-human chimeric antibody against CD۲۰ (۱F۵), we found that the coding sequence of ۱F۵ chimeric heavy-chain gene constructed by SOE-PCR was resistant to polymerase during both PCR and sequencing reactions. Furthermore, we were also unable to analysis some positive transformants by restriction enzyme digestion. Encountering such difficulties to identify the cloned anti-CD۲۰ chimeric heavy-chain gene, we found that the chimeric heavy-chain sequence is highly GC-rich and predicted to form a stable secondary structure. Conclusion: In conclusion, for the first time, we reported several difficulties with production of therapeutic chimeric ۱F۵ anti-CD۲۰ antibody due to a predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly restriction analysis. Our findings provide a probable note for researchers experiencing technical difficulties with construction of chimeric anti-CD۲۰ antibody ۱F۵ gene vectors and also with other genes and molecular biology techniques requiring PCR-based method or restriction enzyme analysis.

کلیدواژه ها:

۱F۵ ، Monoclonal antibody ، Chimeric gene ، SOE-PCR ، Secondary structure