cases annually worldwide Saliva testing can detect potential - PowerPoint PPT Presentation

 OSCC C – malignant tumor with 640,000 new cases annually worldwide  Saliva testing can detect potential biomarkers for OSCC  Elevated level of p53 protein was identified in OSCC patients at different stages of disease

 ~35 monoclonal (mAbs) are approved for therapeutic applications by European Medicines Agency & FDA  Most Abs - chimeric/humanized  Next-ge generation neration Ab Ab fragments gments: antigen- binding fragments (Fab-s), single chain variable fragments (scFv-s), and heavy-chain Ab-s (HcAb-s)

 HcAbs are found in sera of camelids  No constant domain CH1, only variable domains: Vh VhH-s or nanobod bodie ies  Nanobo bodi dies es (Nb) – smallest naturally occurring fragments (around 120 aa)

 Small and soluble  Highly stable  Bind to target with high specificity  Can be conjugated to other proteins without loss of function  Can be expressed and secreted in many organisms including E. coli

Vhhp53 Vhhp53 His His RFP RFP HlyA HlyA

Modified E. Coli having functional type I secretion system will be able to secrete VhHp53 nanobody that contains HlyA secretion signal To purify VhHp53 nanobody secreted by E. coli in order to check its affinity for p53 protein E. Coli derived hcAbs for VhHp53 will help to reduce the time spent on diagnostics of OSCC & to lower the costs

Transforming E. coli with hlyB hlyD yD genes in 1. pVDL9.3 DL9.3 (Cm) m) with pUC5 UC57 7 vector encoding Vhhp hhp53 53 ge genes nes Induce expression of the protein with 1uM 2. IPTG (6, 8 and 10 hours) Measure photometric intensity (Varioscan 3. flash 4.00.53) Purify proteins from the media with Ni/NTA 4. columns SDS PAGE 5. Measure concentration (Nanodrop A280) 6. Check the affinity of obtained nanobodies 7. to p53 protein

0.05 0.04 0.03 control: uninduced culture ty, RFU sity, 0.02 6-hour induction Intensi 10-hour induction 0.01 0 1 2 3 4 Samp mple -0.01

1 st elution: 46.95 ng/ul 2 nd elution: 123.16 ng/ul

 Obtained pure Vh VhH p53  Participated in Interl rlab ab St Study  Next step: to test the affinity of this nanobody for cancer biomarker p53  Futur ure e pla lan: to use this protein-nanobody interaction to design the biosensor for the detection of p53 in saliva samples for OSCC diagnosis

 School of Science and Technology, Nazarbayev University  Technopark, Nazarbayev University  Damira Kanayeva, PhD, Nazarbayev University, Astana, Kazakhstan  Luiz Angel, PhD, CSIC, Madrid, Spain  Rebecca Bocanegra, CSIC, Madrid, Spain  Ana Cuervo, CSIC, Madrid, Spain  Kenneth Alibek, MD, PhD, Nazarbayev University, Astana, Kazakhstan  Gonzalo Hortelano, PhD, Nazarbayev University  Alexander Shustov, PhD, NCB, Astana, Kazakhstan  Sholpan Kauanova, Nazarbayev University, Astana, Kazakhstan  Nurgul Imangali, Nazarbayev University, Astana, Kazakhstan  Madina Shaimerdenova, Nazarbayev University, Astana, Kazakhstan

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