Cloning and Expression of the Recombinant HPV16/18 L1-L2-E7 Polypeptide in Bacterial Expression System

Human papillomaviruses (HPVs) especially types 16 and 18 are known as the major causes of cervical cancer. Among HPV proteins, L1 and L2 capsid proteins, and also E7 oncoprotein are proposed as target antigens for vaccine design. In the recent years, the recombinant multiepitope polypeptides have attracted a special interest. The goal of this study is the design of L1-L2-E7 fusion construct and evaluation of its expression in bacterial expression system. 

In this study, the immunogenic and conserved epitopes of HPV16/18 L1, L2 and E7 proteins were selected using different bioinformatics analyses. After synthesis of the designed L1-L2-E7 fusion sequence in pUC57 cloning vector, its subcloning was performed in pET24a (+) prokaryotic expression vector using EcoRI/ HindIII restriction enzymes. The expression of the recombinant multiepitope polypeptide was done in E.coli Rosetta strain using IPTG inducer, and confirmed by SDS-PAGE and western blotting using anti-His-tag antibody. The expression was optimized under different conditions such as optical density (OD in wavelength of 600 nm), temperature and time after induction, and IPTG concentration.   

The recombinant pET-L1-L2-E7 vector was confirmed by the presence of a clear band (~525 bp) related to the L1-L2-E7 gene on agarose gel after enzyme digestion. The expression of L1-L2-E7 polypeptide in bacteria showed the presence of a clear band (~20 kDa) in SDS-PAGE and western blotting. The best conditions for expression of the recombinant polypeptide were at temperature of 37◦C, optical density of 0.7-0.8, IPTG concentration of 1mM, and time of 16 h after induction.  

The successful expression of the L1-L2-E7 multiepitope polypeptide was performed in bacterial system. In the next step, the recombinant polypeptide will be purified to use as a vaccine candidate.