The production of recombinant proteins Gst L1, E6 and E7 Tag Hpv 16 to be used Luminex test to detect antibodies in the Tunisian population Wi Male Production of recombinant GST proteins L1, E6 and E7 HPV-16 tag to be used in the test for detecting antibodies LUMINEX Tunisian among women with cervical cancer and controls
Achour. M * A, Ben Younes. Ra, Kahla. Sa Kochbati. Lb, Zeghal. Dc Maalej. MB, Zouari. Fc Oueslati. Ra
an environmental laboratory-Immuno-Microbiology and Carcinogenesis (IMEC Unit) Faculty of Sciences Bizerte Tunisia Zarzouna 7021.
b Department of Radiotherapy Cancer Institute Tunisia Azeiz Salah.
c Department of Gynaecology and Obstetrics of the Centre for Maternal and Neonatal Hospital La Rabta Tunisia.
* Corresponding author
Achour Mongia
E-mail: mahaachour2000@yahoo.fr
Tel: 216 72591845
Fax: 216 72590566
SUMMARY:
Some types of human papillomavirus (HPV), mainly HPV types 16 and 18 have been recognized as causative factors for the development of cancer of the cervix. Antibodies against HPV antigens were found associated with the development of cervical cancer. different tests can be used for the detection of antibodies, but they have low levels of sensitivity and specificity. In this work we are interested in preparing recombinant proteins for use in the Luminex technology in order to undergo serological study among women in Tunisia. Thus, HPV 16 L1, E6 and E7 sequences fused to the 3 'end a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (Tag) were isolated from plasmids and inserted into a vector pGEX expression as GST fusion proteins in E. coli. Coding sequences for L1tag, E6tag E7tag and HPV 16 respectively have been mobilized by digestion with enzymes and ligated into plasmids digested downstream of the GST domain. An expression plasmid for the GST tag was constructed by inserting a fragment encoding the epitope tag. The cells of E. coli BL 21 was transformed with pGEX plasmids and grown in Luria Bertani medium containing ampicillin. Recombinant protein expression was induced by adding 0.25 mM isopropyl-?-D-thio-galactoside (IPTG) in the middle. Bacteria were harvested after induction and the bacteria were pelleted resuspended in phosphate buffered saline (PBS) and lysed using a high pressure homogenizer. Lysates were then cleared by centrifugation.
The proteins were verified by migration in sodium dodecyl sulfate (SDS) gel electrophoresis. The data showed they were stable for the detection and the lysates were stored at -20 ° C to be used in Luminex for the detection of antibodies among Tunisian women. This test showed that seropositivity to different antigens differs in the treatment group and differences between cases and controls were significant (P <0.001). In addition, an elevated percentage of positivity was found for E7 (61%) against 44% with only 21% for antigens E6 and L1, respectively, and the intensity of the immune response towards the end of the L1 antigen and early antigens E6 and E7 were different.
Keywords: tag GST - E coli - HPV 16 - L1 - E6 - E7 - LUMINEX
INTRODUCTION:
Globally, the human papillomavirus (HPV) is estimated to cause nearly half a million cases and 270,000 deaths from cancer of the cervix, which is more than 2.5 million years of life lost (YLL) per year (Sue et al., 2007).
HPV type 16 (and to a lesser degree HPV type 18) is linked to rare cancers, namely cancer of the vulva, vagina, penis, anus, oropharynx and larynx. Effective prophylactic vaccines have been developed (Dillner et al., 2007). Molecule.
Posted on July 29, 2011.
