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

• ccurring 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

• rganisms including E. coli

His

RFP

HlyA

Vhhp53

His

RFP

HlyA

Vhhp53

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

• rder 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

1.

Transforming E. coli with hlyB hlyD yD genes in pVDL9.3 DL9.3 (Cm) m) with pUC5 UC57 7 vector encoding Vhhp hhp53 53 ge genes nes

2.

Induce expression of the protein with 1uM IPTG (6, 8 and 10 hours)

3.

Measure photometric intensity (Varioscan flash 4.00.53)

4.

Purify proteins from the media with Ni/NTA columns

5.

SDS PAGE

6.

Measure concentration (Nanodrop A280)

7.

Check the affinity of obtained nanobodies to p53 protein

• 0.01

0.01 0.02 0.03 0.04 0.05 1 2 3 4

Intensi sity, ty, RFU

Samp mple

control: uninduced culture 6-hour induction 10-hour induction

1st elution: 46.95 ng/ul 2nd 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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