Construction and cloning of a recombinant expression vector containing human Cd20 Gene for antibody therapy in Non-Hodgkin Lymphoma

Non-Hodgkin lymphoma (NHL) is a cancer that starts in lymphocytes. The main treatment for NHL is chemotherapy and radiation. Today immunotherapy is a promising therapeutic approach in the treatment of a variety cancers which is high specific unlike previous methods. Antibodies do not penetrate effectively into tumore tissues because of their large size. Whereas the small size of nanobodies (Camelid single-domain antibodies) allow them to efficiently enter into tissues and bind to the epitopes. Human CD20 over expression in the B-cell lymphoma makes this antigen as a validated target for immunotherapy. One major problem in production of full length CD20 is aggregation and misfolding. Therefore, production of a polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20.
The fragment consisting of human CD20 extra membrane loop and hinge and Fc of camelid IgG was constructed. Then it was facilitated by HindIII and XhoI restriction enzyme sites and fused to the 6× His tag for purifiction. The stop codon was engineered in the terminal sequence. The engineered coding sequence was synthesized by Generay Company and then inserted into the pcDNA3.1 () vector to obtain recombinant expression vector. The accuracy of ligation reaction was confirmed by colony PCR, sequencing and digestion.
The colony PCR result showed 1132 kb fragment. The results of digestion and sequencing showed that the protein was what we had hoped to acquire.
We have obtained the recombinant expression vector inorder to express in mammalian cell which can be used to produce novel anti-human CD20 monoclonal antibody in future.