Production and Characterization of High-Affinity Antibodies Reactive Towards HEp-2 Cells Nuclei by Injection of an In Silico Designed Recombinant Truncated Nuclear Mitotic Apparatus Protein
Abstract The Nuclear Mitotic Apparatus protein (NuMA) is a nuclear protein that plays critical role in the mitosis as an organizer of the mitotic spindles and is a prevalent protein in malignant urothelial cells which makes it an appropriate marker for bladder cancer early detection. In this study, NuMA protein sequence was obtained from Genbank based on literature studies. Immunodominant epitopes which are located between residues 1 and 300 were reverse translated and optimized for expression in E. coli cells. Final sequence was then synthesized, cloned into pGS21a vector and expressed in BL21 cells. The expressed protein was examined by SDS-PAGE and also Western blotting to confirm expression by using anti-His antibody-HRP. The protein was then purified using nickel chromatography column and 18 mg of purified truncated NuMA was obtained from 1 l of bacterial culture. ELISA was performed to evaluate the reactivity of commercial anti-NuMA antibody towards truncated protein. Finally, the antigen was injected and polyclonal antibody was produced in rabbits. The immunoglobulins of antiserum were precipitated by ammonium sulfate and purified by DEAE–Cellulose chromatography column. To evaluate antibody accuracy, it was tested on HEp-2 cells with permeable nucleus membranes fixed on slides. The antibody detected NuMA protein in the nuclei of HEp-2 cells, which means it can have diagnostic value in the bladder cancer patients. The truncated NuMA is a suitable protein as a single multiepitope antigen that further may be used for studies to develop assays for early diagnosis of bladder cancer..
| Medienart: |
Artikel |
|---|
| Erscheinungsjahr: |
2018 |
|---|---|
| Erschienen: |
2018 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:25 |
|---|---|
| Enthalten in: |
International journal of peptide research and therapeutics - 25(2018), 2 vom: 01. Juni, Seite 727-738 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Soukhtehzari, Sepideh [VerfasserIn] |
|---|
| Links: |
Volltext [lizenzpflichtig] |
|---|
| BKL: | |
|---|---|
| Themen: |
| Anmerkungen: |
© Springer Science+Business Media, LLC, part of Springer Nature 2018 |
|---|
| doi: |
10.1007/s10989-018-9719-4 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
OLC2067155253 |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | OLC2067155253 | ||
| 003 | DE-627 | ||
| 005 | 20230510063330.0 | ||
| 007 | tu | ||
| 008 | 200820s2018 xx ||||| 00| ||eng c | ||
| 024 | 7 | |a 10.1007/s10989-018-9719-4 |2 doi | |
| 035 | |a (DE-627)OLC2067155253 | ||
| 035 | |a (DE-He213)s10989-018-9719-4-p | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 082 | 0 | 4 | |a 540 |q VZ |
| 084 | |a 35.76$jAminosäuren$jPeptide$jEiweiße$XBiochemie |2 bkl | ||
| 100 | 1 | |a Soukhtehzari, Sepideh |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Production and Characterization of High-Affinity Antibodies Reactive Towards HEp-2 Cells Nuclei by Injection of an In Silico Designed Recombinant Truncated Nuclear Mitotic Apparatus Protein |
| 264 | 1 | |c 2018 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ohne Hilfsmittel zu benutzen |b n |2 rdamedia | ||
| 338 | |a Band |b nc |2 rdacarrier | ||
| 500 | |a © Springer Science+Business Media, LLC, part of Springer Nature 2018 | ||
| 520 | |a Abstract The Nuclear Mitotic Apparatus protein (NuMA) is a nuclear protein that plays critical role in the mitosis as an organizer of the mitotic spindles and is a prevalent protein in malignant urothelial cells which makes it an appropriate marker for bladder cancer early detection. In this study, NuMA protein sequence was obtained from Genbank based on literature studies. Immunodominant epitopes which are located between residues 1 and 300 were reverse translated and optimized for expression in E. coli cells. Final sequence was then synthesized, cloned into pGS21a vector and expressed in BL21 cells. The expressed protein was examined by SDS-PAGE and also Western blotting to confirm expression by using anti-His antibody-HRP. The protein was then purified using nickel chromatography column and 18 mg of purified truncated NuMA was obtained from 1 l of bacterial culture. ELISA was performed to evaluate the reactivity of commercial anti-NuMA antibody towards truncated protein. Finally, the antigen was injected and polyclonal antibody was produced in rabbits. The immunoglobulins of antiserum were precipitated by ammonium sulfate and purified by DEAE–Cellulose chromatography column. To evaluate antibody accuracy, it was tested on HEp-2 cells with permeable nucleus membranes fixed on slides. The antibody detected NuMA protein in the nuclei of HEp-2 cells, which means it can have diagnostic value in the bladder cancer patients. The truncated NuMA is a suitable protein as a single multiepitope antigen that further may be used for studies to develop assays for early diagnosis of bladder cancer. | ||
| 650 | 4 | |a In silico design | |
| 650 | 4 | |a NuMA antibody | |
| 650 | 4 | |a Truncated NuMA | |
| 700 | 1 | |a Rasaee, Mohammad Javad |4 aut | |
| 700 | 1 | |a Javanmardi, Masoud |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t International journal of peptide research and therapeutics |d Springer Netherlands, 2005 |g 25(2018), 2 vom: 01. Juni, Seite 727-738 |w (DE-627)490328148 |w (DE-600)2193110-0 |x 1573-3149 |7 nnns |
| 773 | 1 | 8 | |g volume:25 |g year:2018 |g number:2 |g day:01 |g month:06 |g pages:727-738 |
| 856 | 4 | 1 | |u https://doi.org/10.1007/s10989-018-9719-4 |z lizenzpflichtig |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a SYSFLAG_A | ||
| 912 | |a GBV_OLC | ||
| 912 | |a SSG-OLC-PHA | ||
| 912 | |a SSG-OLC-DE-84 | ||
| 936 | b | k | |a 35.76$jAminosäuren$jPeptide$jEiweiße$XBiochemie |q VZ |0 181571129 |0 (DE-625)181571129 |
| 951 | |a AR | ||
| 952 | |d 25 |j 2018 |e 2 |b 01 |c 06 |h 727-738 | ||