a- Laboratory of Environmental Immuno-Microbiology and cancerogenesis (IMEC Unit) Faculty of Sciences Bizerta 7021 Zarzouna Tunisia.
b- Service of Radiotherapy Institute of Cancer Salah Azeiz Tunisia.
c- Service of Gynecology- obstetrics Center of Maternity and Neonatalogy Hospital La Rabta Tunisia.
Certain types of Human Papillomavirus (HPVs), mainly HPV types 16 and 18 have been recognised as major etiological factors for the development of cervical cancer. Antibodies against HPV antigens have been found to be associated with cervical cancer evolution. Different assays can be used for antibody detection but they have low levels of sensitivity and specificity. In the present work we are interested to prepare recombinant proteins to be used in LUMINEX technology in order to undergo serological study in Tunisian female population. So, HPV types 16 L1, E6 and E7 sequences fused to their 3'-end to a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (tag) were isolated from plasmids and inserted into a pGEX vector for expression as GST fusion proteins in E.coli. Coding sequences for L1tag, E6tag and E7tag of HPV 16 respectively were mobilized by digestion with enzymes and ligated into digested plasmids downstream of the GST domain. An expression plasmid for GST tag was constructed by inserting a fragment coding for the tag epitope. Cells of E.coli BL 21 were transformed with the pGEX plasmids and grown in Luria Bertani medium containing the ampicillin. Recombinant proteins expression was induced by adding 0.25 mM isopropyl-?-D-thio-galactoside (IPTG) to the medium. The bacteria were harvested after induction and pelleted bacteria were resuspended in phosphate buffered saline (PBS) and lysed using a high- pressure homogenizer. Lysates were then cleared by centrifugation.
Proteins were verified by migration in sodium dodecyl sulphate (SDS) gel electrophoresis. Data showed that they were stable for detection and these lysates have been conserved at -20°C to be used in Luminex for detection of antibodies in female Tunisian population. This assay showed that the sero- positivity toward the different antigens differs upon the group studied and differences between cases and controls were significant (P<0.001). In addition, elevated percentage of positivity was found for E7 (61 %) versus 44 % and only 21 % for E6 and L1 antigens respectively and the intensity of the antibody response toward the late antigen L1 and the early antigens E6 and E7 were different.
On a global level, human papillomavirus (HPV) is estimated to cause almost half a million cases and more than 270,000 deaths from cervical cancer, corresponding to more than 2.5 million years of life lost (YLL) annually (Sue et al., 2007).
HPV type 16 (and to a lesser degree HPV type 18) is linked with more rare cancers, namely cancer of the vulva, vagina, penis, anus, oropharynx and larynx. Effective prophylactic vaccines have been developed (Dillner et al., 2007). Molecular epidemiological studies have demonstrated that specific subtypes of HPV are associated with cervical cancer (Castle and Giuliano, 2003; Dillner and Brown, 2004).
The HPV group of viruses today consists of more than 100 completely characterized types. Partial sequences of additional isolates indicate that at least another 100 HPVs exist. Of these, 15 genital HPVs are established as oncogenic in humans. HPV type 16 is by far the most important virus, accounting for more than 50% of all cervical cancers. HPV16 is even more dominating as an etiology of the noncervical HPV-associated cancers (Dillner, 2005). HPV serology is complex for several reasons. Different types of HPV can infect the epithelia of skin or mucosa and induce proliferative diseases. HPV antibodies are type specific. Those targeting the major viral capsid protein L1 are markers of infection, and those targeting the viral oncoproteins E6 and E7 are markers for HPV-associated cancer (Waterboer et al., 2005). Conventional serologic methods such as ELISA allow the analysis of sera for antibodies to only 1 antigen per well. Previous studies have determined antibodies against early antigens as E6 protein by ELISA methods that use small, linear epitopes of the proteins but sometimes they show low sensitivities and specificities. To improve the immunologic method, other approaches have been advanced like radioimmunoprecipitation assays (RIPAs) with whole native proteins and sandwich ELISAs with full-length (Meschede et al., 1998). Today, HPV serology is performed mostly in a limited number of laboratories, but it is likely to become widely used in clinical laboratories in the post-HPV vaccination sera. Previous studies showed that the LUMINEX method constitutes an attractive method for HPV serology in high-throughput laboratories (Waterboer et al., 2006).
In the present report, viral antigens were expressed with pGEX vectors in Escherichia coli as double fusion proteins with N-terminal GST and a C-terminal peptide (tag) consisting of the 11 C-terminal amino acids from the large T antigen of simian virus 40. The protein concentrations of the cleared lysates have been determined using the Bradford-reagent and the characterization of the full-length recombinant proteins was verified by coomassie-stained SDS-PAGE. Furthermore, the titration of antigen lysates was done using mouse anti-tag antibodies. These antigens were then used in LUMINEX assay for antibody detection in Tunisian female population.
A modified pGEX vector was constructed for expression of GST fusion protein with an additional C-terminal fusion tag in E.coli. HPV 16 L1 coding sequence lacking the 10 N-terminal residues was amplified by polymerase chain reaction PCR with SmaI/SalI ends and inserted into pGEX4T3tag opened by EcoRI digestion.
E.coli BL21 cells transformed with the pGEX plasmids were grown at room temperature in Luria Bertani medium containing 1mM ampicillin; At an OD600 of 0.3 recombinant protein expression was induced by adding 0.25 mM isopropyl-?-D-thio-galactoside (IPTG) to the medium. The bacteria were harvested by centrifugation 15 h after induction. Pelleted bacteria were resuspended in 40 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA (ethylenediaminetetraaceticacid) and 2 mM DTT (dithiothreitol) supplemented with complete protease inhibitor cocktail and lysed using a high pressure homogenizer. ATP (adenosine triphosphate) and MgCl2 were added to final concentration of 2 mM and 5 mM respectively (Sehr et al., 2001).
HPV 16 E6 and E7
HPV type 16 E6 and 16 E7 coding sequence fused at its 3'-end in frame to a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (tag) is isolated from bluescript plasmid and inserted into a pGEX vector for expression as GST fusion protein in E.coli. These coding sequences for E6tag and E7tag were mobilized by digestion and ligated into digested plasmid downstream of the GST domain.
Then, E.coli BL 21 cells transformed with the pGEX plasmids were grown and induction protein expression was induced as previously described for L1. The bacteria were harvested 6h after induction by centrifugation. Pelleted bacteria were resuspended in PBS containing 2 mM DTT, 1% Triton X-100, and complete protease inhibitor cocktail and lysed with the high-pressure homogenizer. Lysates were then cleared by centrifugation and stored in aliquots at -20°C (Sehr et al., 2002).
EAS (Electronic Article Surveillance), also known as Electronic Article Surveillance (Pirates) system is one of the major commodity security measures currently widely used in the retail industry. EAS in the mid-1960s in the United States come, first applied to the garment industry, has now been extended to more than 80 countries and regions worldwide, applications are extended to department stores, supermarkets, book a variety of industries, especially the mini tag application of large supermarkets (storage) of fully developed.
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| EAS tag |
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