2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's
| Format: | General Document |
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| building | INTELEK Repository |
| collection | Online Access |
| collectionurl | https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection3 |
| copyright | Copyright©PWB2025 |
| country | Malaysia |
| date | 2023-08-27 |
| format | General Document |
| id | 15409 |
| institution | UniSZA |
| internalnotes | Sila masukkan subject wajib Dissertations, Academic untuk semua tesis.. Terima kasih |
| originalfilename | CLONING, SITE.DIRECTED MUTAGENESIS AND EXPRESSION OF STNGLE CHAIN FRAGMENT VARIABLE (SCFV) OF C3A8 HYBRIDOMA AGAINST BREAST MCF-7 CARCINOMA CELLS IN PICHIA PASTOR'S.pdf |
| person | Syaidatul Najiah Zakaria |
| recordtype | oai_dc |
| resourceurl | https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15409 |
| sourcemedia | Server storage Scanned document |
| spelling | 15409 https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15409 https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection3 General Document Malaysia Library Staff (Top Management) Library Staff (Management) Library Staff (Support) Terengganu Faculty of Bio-resources & Food Industry English application/pdf 1.5 Server storage Scanned document Universiti Sultan Zainal Abidin UniSZA Private Access Universiti Sultan Zainal Abidin SAMBox 2.4.24; modified using iTextSharp™ 5.5.10 ©2000-2016 iText Group NV (AGPL-version) 2023-08-27 170 CLONING, SITE.DIRECTED MUTAGENESIS AND EXPRESSION OF STNGLE CHAIN FRAGMENT VARIABLE (SCFV) OF C3A8 HYBRIDOMA AGAINST BREAST MCF-7 CARCINOMA CELLS IN PICHIA PASTOR'S.pdf Syaidatul Najiah Zakaria 2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's Copyright©PWB2025 The single chain fragment variable (scFv) antibodies of C3AB hybridoma clone secreted lgM monoclonal antibody against breast cancer cells was constructed earlier by using phage display technology. The recombinant plasmid containing gene of anti-MCF-7 single chain fragment variable antibody was isolated from clone f G1, Escherichia coli and the presence of the gene was confirmed through PCR amplification, restriction digestion and sequencing analysis. The gene was 100% similar to the anti-MCF-7 scFv gene in the GeneBank database (EU980594). ln this study, the anti-MCF-7 scFv antibodies gene was cloned and expressed in the pichia pastorls expression system as an alternative expression system to the E. col1. The gene was cloned into the pPlCZoA expression vector and the new restriction sites, EcoRl_anti-McF-7 scFv_Not I construct were introduced. The pPlCZoA_EcoRl_anti-MCF-7 scFv_Nof I construct was transformed into p. pastons strain X-33 and two positive clones were selected (clone 2Al and 246). However, the scFv proteins of these two clones were failed to appear when analysed using SDS-PAGE. The failure was due to the presence of the stop codon at the position nine in the framework region (FR). The gene was re-engineered to a functional anti-MCF-7 scFv gene through site-directed mutagenesis (SDM) by changing the stop codon to serine (S) and modifies the restriction sites from EcoRl, Not I to C/a l, Xba l.fhe mutated C/a l_anti-MCF 7 scFv_Xba I construct was cloned in pPlCZoC expression vector and transformed into P pasforis strain X-33. A 34 kDa anti-MCF-7 scFv antibody band was successfully expressed and observed on the SDS-pAGE gel. The expressed anti-MCF-7 scFv antibodies were characterized by immunoblotting where the band was detected using His{ag peptide which was fused at the carboxyl terminus of the scFv. ln conclusion, the functional scFv antibodies were successfully cloned and expressed in p pastor's through site-directed mutagenesis. Dissertations, Academic Sila masukkan subject wajib Dissertations, Academic untuk semua tesis.. Terima kasih Cloning (Molecular biology) Single Chain Fragment Variable (scFv) C3A8 Hybridoma MCF-7 Breast Carcinoma Cells Thesis |
| spellingShingle | 2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's |
| state | Terengganu |
| subject | Dissertations, Academic Cloning (Molecular biology) |
| summary | The single chain fragment variable (scFv) antibodies of C3AB hybridoma clone secreted lgM monoclonal antibody against breast cancer cells was constructed earlier by using phage display technology. The recombinant plasmid containing gene of anti-MCF-7 single chain fragment variable antibody was isolated from clone f G1, Escherichia coli and the presence of the gene was confirmed through PCR amplification, restriction digestion and sequencing analysis. The gene was 100% similar to the anti-MCF-7 scFv gene in the GeneBank database (EU980594). ln this study, the anti-MCF-7 scFv antibodies gene was cloned and expressed in the pichia pastorls expression system as an alternative expression system to the E. col1. The gene was cloned into the pPlCZoA expression vector and the new restriction sites, EcoRl_anti-McF-7 scFv_Not I construct were introduced. The pPlCZoA_EcoRl_anti-MCF-7 scFv_Nof I construct was transformed into p. pastons strain X-33 and two positive clones were selected (clone 2Al and 246). However, the scFv proteins of these two clones were failed to appear when analysed using SDS-PAGE. The failure was due to the presence of the stop codon at the position nine in the framework region (FR). The gene was re-engineered to a functional anti-MCF-7 scFv gene through site-directed mutagenesis (SDM) by changing the stop codon to serine (S) and modifies the restriction sites from EcoRl, Not I to C/a l, Xba l.fhe mutated C/a l_anti-MCF 7 scFv_Xba I construct was cloned in pPlCZoC expression vector and transformed into P pasforis strain X-33. A 34 kDa anti-MCF-7 scFv antibody band was successfully expressed and observed on the SDS-pAGE gel. The expressed anti-MCF-7 scFv antibodies were characterized by immunoblotting where the band was detected using His{ag peptide which was fused at the carboxyl terminus of the scFv. ln conclusion, the functional scFv antibodies were successfully cloned and expressed in p pastor's through site-directed mutagenesis. |
| title | 2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's |
| title_full | 2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's |
| title_fullStr | 2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's |
| title_full_unstemmed | 2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's |
| title_short | 2012_Cloning, Site-Directed Mutagenesis and Expression of Single Chain Fragment Variable (scFv) of C3A8 Hybridoma against Breast MCF-7 Carcinoma Cells in Pichia Pastor's |
| title_sort | 2012_cloning, site-directed mutagenesis and expression of single chain fragment variable (scfv) of c3a8 hybridoma against breast mcf-7 carcinoma cells in pichia pastor's |