Cervical Cancer HPV16E6 Antigen/HBD-2 Chimeric Nucleic Acid Vaccine And Its Expression In Mammalian Cells And Mice

Antimicrobial polypeptides are important components of the innate immunity. They are thought to be a key factor of host against infection owing to their broad-spectrum antimicrobial activity. Defensins, the earliestly characterized family of amtimicrobial polypeptides of mammalian cells , are small cationic, cysteine-rich peptides and are composed of α-, β-, θ-defensins classes which differ from one another by the connectivity of their six cysteine residues. Up to now, three β- defensins of epithelial origin, HBD1, HBD2, HBD3, have been identified and characterized in humans. The HBD2 is composed of 41 amino acids with six-cysteine residues. The HBD2 identifies to date have the capability to kill a particular spectrum of bacteria, fungi or some enveloped viruses in vitro and is generally considered to be direct effector of innate antimicrobial immunity. HBD2 also acts as regulators of innate immunity. The chemotactic effect of HBD2 on immature DC cells is mediated by human CC chemokine receptor 6(CCR6). Such 'definsin-Ag' might, by definsin-receptor-mediated internalization, deliver Ags to immatureDCs, thereby facilitating the uptake of Ags. Secondly, HBD2 might also promote the maturation of DCs either directly or indirectly through the induction of TNF and IL-1. Finally, it might also facilitate the recruitment of memory T cells to the infected tissue, thus contributing to the effector phase of adaptive immune response. On the basis of its immunoregulatory roles, it might also be good candidate for the development of vaccine adjuvant because it has been shown to enhance adaptive immunity markedly.Extensive epidemiologic data have demonstrated a strongly association of high-risk HPV16 with cervical carcinoma. Because its early open reading frame protein E6 is selective retained and expressed in cervical tumors, E6 is attractive targets for immunotherapies. The E6 protein has the transforming activity. The gene fragment of C-terminal region of HPV16E6, whose the transforming activity has been eliminated and antigenicity is reserved, was generally used as effective target antigen of inducing tumor immunity.In order to enhance the immunogenicity of C-terminal fragment of HPV16E6, using HBD-2 as an adjuvant molecule, we constructed the chimeric HBD-2/ HPV16E6 C-terminal fragment eukaryiotic expressive plasimid. According to the cDNA sequence of HPV16E6 gene and HBD2 from the Genbank (No. NC001526, AF071216), we designed the specific primers to amplify the HPV16E6 and HBD2 segment. Using cloning technique, the cDNA fragments of HBD2 gene and HPV16E6C gene were constructed into eukaryotic expression plasmid pcDNA3.1.The inserted target genes in the plasmid were verified by nucleotide sequecing. COS7 cell line was transfected with the recombinant plasmid pcDNA3.1/HBD2-HPV16E6C by using lipofectamine reagent. RT-PCR and immunohistochemistry wereperformed to identify the expression of fusion gene HBD2-HPV16E6C in the transfected cells. Mice were immunized i.m. The results indicated that the expression of HBD-2, HPV16E6C, and HBD-2/HPV16E6C chimera genes could be detected at mRNA and protein level in transfected cells and in the recombinant injected mouse muscular tissues. This study provides a good basis for further study on HPV16E6C DNA vaccine.