بیوسنسور DNA الکتروشیمیایی برای شناسایی ژن E6 ویروس پاپیلوم انسانی داخل پلاسمید نوترکیب Electrochemical DNA biosensor for the detection of human papillomavirus E6 gene inserted in recombinant plasmid

Abstract In the current study, we describe a novel, simple, inexpensive, sensitive, specific, stable and label-free electrochemical DNA biosensor used to identify a target gene cloned into a plasmid. The biosensor was designed with a 23-mer oligonucleotide of guanine-free, which was immobilized on the pencil graphite electrode (PGE) for E6 gene detection from human papillomavirus 16 type (HPV16). The E6 gene was used due to its clinical importance. The optimal probe concentration was obtained in 500 nM. The hybridization detection showed a good linearity in the range of 40–۵,۰۰۰ pg/lL with a detection limit of 16 pg/lL. The electrochemical method showed higher sensitivity and specificity when compared with the agarose gel electrophoresis assay. This technology could be postulated as a new and attractive alternative for cloning analysis in plasmids. ۱٫ Introduction There are over 160 known types of human papillomavirus (HPV) (Burk et al., 2013), of which 40 can infect the anogenital epithelium and of these 15 are considered oncogenic (Lin et al., 2010). The HPV 16 and 18, classified as high-risk types (HR), are responsible for approximately 60–۸۰% of cervical cancer occurrence worldwide (Carter et al., 2011; Hendry et al., 2013). Several studies show that HPV encodes two powerful oncogenes, E6 and E7. These oncogenes are constantly expressed in the HR-HPV and are responsible for the malignant transformation of cervical cancer (Azam and Shams-ul-Islam, 2010; Boccardo et al., 2010; Carter et al., 2011; Stanley, 2010; Vici et al., 2014). Therefore, the E6 and E7 genes represent the ideal targets for development of therapeutic vaccines, which potentially eliminate pre-existing lesions and malignant tumors by generating cellular immunity against HPV-infected cells (Huang et al., 2010; Kawana et al., 2012; Nieto and Salvetti, 2014). Progress in the molecular cloning techniques has enabled the relatively quick, easy and cheap manufacture of recombinant vector vaccines (Huang et al., 2010; Hung et al., 2008). Recombinant vector vaccines have many advantages over conventional vaccines and may provide a technological solution for microorganisms that have difficulty growing in cell culture or animal models, like HPV (Ferraro et al., 2011; Lin et al., 2010; Ma et al., 2010). The most popular technique used for molecular cloning analysis is the electrophoresis method in agarose gels. However, this technique requires well-trained personnel and is time-consuming (Chang et al., 2013; Teles and Fonseca, 2008). DNA biosensors are commonly employed to detect a specific DNA sequence (Pei et al., 2013; Tosar et al., 2010), but less explored for cloning analysis. In the current study we describe a novel, simple, inexpensive, stable and label-free DNA electrochemical biosensor for the detection of HPV 16 E6 gene cloned in the expression vector. ۲٫ Materials and methods ۲٫۱٫ Materials Escherichia coli DH5a (Invitrogen – USA) and pGEM-T Easy (pGEM-T) (Promega – USA) were used as host and cloning vectors, respectively. Bacterial growth was performed in a Luria–Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L, pH 7.0), supplemented with 100 mg/ L ampicillin, 160 lg/mL X-gal and 0.5 mM IPTG (Isopropyl b-D-1-thiogalactopyranoside). T4 DNA ligase, DNA size marker and EcoRI, XbaI and ApaI restriction enzymes were purchased from Invitrogen – USA. DNA polymerase and QIAquick PCR Purification Kit were supplied from Clontech Company – USA and QIAgen – Germany, respectively. Pencil lead (type 4B), commonly composed of natural graphite, a polymeric binder and clay in different percentages, was used as pencil graphite electrode (PGE). The 23-mer, guanine-free oligonucleotide (50 -ATI CAC CAA AAI AIA ACT ICA AT-30 , purchased from Integrated DNA Technologies – USA), correspondent to the sense strand of the HPV 16 E6 gene, was employed as HPV probe. The oligonucleotide solutions and dilute solutions of the plasmids were prepared with 0.5 M acetate buffer (pH 5.0) and kept frozen.