|Year : 2014 | Volume
| Issue : 2 | Page : 265-273
Construction, expression and characterisation of a single chain variable fragment in the Escherichia coli periplasmic that recognise MCF-7 breast cancer cell line
Elham Omer Mahgoub, Ahmed Kamal Bolad
Department of Microbiology and Unit of Immunology, Alneelain University Faculty of Medicine, Alneelain Medical Research Center, Khartoum, Sudan
|Date of Web Publication||14-Jul-2014|
Elham Omer Mahgoub
Department of Microbiology and Unit of Immunology, Alneelain University, Faculty of Medicine, Alneelain Medical Research Center, Khartoum
Source of Support: None, Conflict of Interest: None
Background: A functional single-chain fragment variable (scFv) recognizing the MCF-7 breast cancer carcinoma cell line was constructed from the C3A8 hybridoma using phage display technology.
Aim of Study: This study was conducted to evaluate the binding activity of scFv antibody recognise MCF-7 breast cancer cells carcinoma, the scfv antibody constructed and expressed in Escherichia coli periplasmic.
Materials and Methods: The scFv coding sequence was cloned in frame with the pIII phage coat protein. The signal sequence included in the C terminus directed the expression of the scFv in the Escherichia coli periplasm. Following several rounds of biopanning, colonies that expressed a scFv that recognized MCF-7 cells in Western blots, ELISAs, and flow cytometry test were isolated.
Results: A 750-bp scFv gene was successfully isolated. Cloning and two rounds of biopanning isolated the candidate with the highest activity (clone B7), as screened by ELISA. Following poly-acrylamide gel electrophoresis (SDS-PAGE) of the purified product, a 32-kDa band was observed. A similar-sized band was observed following Western blot analysis with an E tag-specific antibody. Binding reactivity of scFv antibody with MCF cells was determined using indirect ELISA and compared with monoclonal antibodies' reactivity. Also, flow cytometry was useful in further characterization to the binding reactivity of scFv antibody with MCF-7 cells.
Conclusions: The recombinant antibody technology used in this study is a rapid and effective approach that will aid in the development of the next generation of immunodiagnostic reagents.
结果：750 bp scFv基因被成功分离。筛选分离具有最高的活性克隆B7。聚丙烯酰胺凝胶电泳（SDS PAGE）纯化后的产物观察到的是一个32 kDa带。Western印迹观察到同样大小的蛋白。与MCF细胞单链抗体的结合反应，用间接ELISA测定，并与单克隆抗体的反应性的比较。同时，流式细胞仪在进一步的MCF 7细胞的单链抗体的结合反应的特征描述上也是有用的。
Keywords: Indirect ELISA, phage display technology, preiplasmic, single-chain fragment variable
|How to cite this article:|
Mahgoub EO, Bolad AK. Construction, expression and characterisation of a single chain variable fragment in the Escherichia coli periplasmic that recognise MCF-7 breast cancer cell line. J Can Res Ther 2014;10:265-73
|How to cite this URL:|
Mahgoub EO, Bolad AK. Construction, expression and characterisation of a single chain variable fragment in the Escherichia coli periplasmic that recognise MCF-7 breast cancer cell line. J Can Res Ther [serial online] 2014 [cited 2020 Oct 29];10:265-73. Available from: https://www.cancerjournal.net/text.asp?2014/10/2/265/136551
| > Introduction|| |
Using phage display technology, high-affinity antibodies with unique specificities can be isolated, allowing for the development of potent immuno-therapies for the treatment of human diseases, including autoimmune disease and cancer. During this technique, a fusion protein is generated in which the coding sequence of a single-chain fragment variable (scFv) fragment derived from the variable (V) regions of an antibody is fused to the amino terminus of the phage minor coat protein pIII. The fusion protein is displayed using a phage vector that is based on the genome of fd-tet.  In the phage vector, the coding sequence of the scFv fragment is cloned in frame with gene III and downstream of the signal sequence, which normally directs the protein to the periplasm,  demonstrated that it was possible to use the filamentous bacteriophage fd as a tool to select an antibody fragment from a large population of non-binding proteins using a technique known as biopanning, and scFv fragments were the first proteins to be successfully displayed on the surface of a phage.  In the fusion protein, the V H and V L domains fold correctly as they are stabilized by an intra-molecular disulphide bridge, and they pair to form a functional scFv fragment.
The use of display vectors and in vitro selection technologies has transformed the way in which ligands, such as antibodies and peptides, have been identified. A variety of tags that are appended to the antibody fragment for their detection have been described, including the myc-derived tag recognized by the antibody 10 9 ,  the Flag sequence and the E tag sequence. This strategy allows for the use of unpurified phage antibodies or antibody fragments that are present in crude supernatants or periplasmic extracts for screening assays. Additionally, the presence of the tag allows for the purification of the tagged phage antibodies using affinity chromatography with tag-specific antibodies. ,
The design of panels of ligands from scratch is possible, and phage displayed-based technologies can aid in the selection of ligands with the desired (biological) properties. However, while phage display has considerable advantages over hybridoma technology, there are several drawbacks to phage display.  For example, the required transformation step limits the library size to approximately 10 10 , which necessitates a substantial labor investment, as detailed by Dower and Cwirla.  Furthermore, selection in the context of the host environment cannot be completely avoided, possibly resulting in the loss of potential candidates due to a growth disadvantage or even toxicity in Escherichia coli. Importantly, however, the expression of scFv fragments in the Escherichia coli periplasm allows for rapid assessment of the antigen-binding properties, and the produced antibody can be affinity-purified on immobilized antigen.
Recently, a wide array of antibodies has been selected from naïve, un-immunized organisms using phage display. Phage display provides novel peptides that bind protein targets with high affinity and specificity, and most marketed, peptide-based drugs are receptor agonists derived from natural peptides. Additionally, novel strategies have been developed for measuring inhibiting receptor-ligand interactions in an attempt to identify antagonists.  The phage display system developed by Krebber  was modified in this work to generate scFv genes from monoclonal Immunoglobulin M (IgM) molecules. The resulting scFv was expressed in the bacterial periplasm and specifically recognized the breast cancer cell line MCF-7. Furthermore, the E tag monoclonal antibody specifically recognized the expressed scFv, and indirect The was used to characterize the affinity of the expressed scFv. Finally, the affinity of the scFv was compared with the parental C3A8 monoclonal antibody produced from the hybridoma. Furthermore, flow cytometry was used for further characterization to the affinity of the expressed scFv protein.
| > Materials and methods|| |
In the materials, the Recombinant Phage Antibody System (including the mRNA Isolation Kit, Mouse ScFv Module, Expression Module, and Detection Module), anti-E tag antibody, mid-phage plasmid (pCANTAB5) Sequencing Primer Set, and the Sfi I and Not I are restriction enzymes were purchased from Pharmacia Biotech (Uppsala, Sweden). The Taq DyeDeoxy Terminator Cycle Sequencing Kit for DNA sequencing and Taq DNA polymerase were from Applied Bio-systems (Foster City, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM was from Kirkegaard and Perry Laboratories, USA). All chemicals and reagents were from Sigma (USA). The antibody cloning and phage display system of Krebber et al.  was used with the following modifications.
In reverse transcriptase-PCR amplification of scFv genes, the hybridoma cell line C3A8 was obtained, and total RNA was extracted from 1 × 10 6 C3A8 cells using the Quick Prep mRNA Purification Kit (Pharmacia, Sweden). Reverse transcription was carried out using the First-Strand cDNA Synthesis Kit (Pharmacia, Piscataway, N.J.) with 5 μg of total RNA in a reaction volume of 33 μl, as recommended by the manufacturer. Two separate reactions were performed to reverse-transcribe the light chain and heavy chain with the primers specified by Krebber et al.  The cDNAs coding for the V regions of the heavy and light chains (V H and V L , respectively) were then amplified using PCR with a set of primers that were included in the Mouse ScFv Module of Recombinant Phage Antibody System (Pharmacia Biotech, Sweden). The PCR amplification was run for 30 cycles (94°C for 1 min; 55°C for 2 min; 72°C for 2 min). The amplified V H and V L fragments were purified separately via agarose gel electrophoresis to remove primers from the amplification products.
The purified V H and V L cDNAs (112 fmol of each) were each assembled into a single gene using a DNA linker fragment (32 fmol) that codes for GGGGSGGGGSGGGGS, which connected the two cDNAs in the correct reading frame. The assembly PCR was run for seven cycles (94°C for 1 min; 63°C for 4 min), and the reaction products were analyzed on a 1% agarose gel. The assembled fragment was amplified using two oligonucleotide primers with either an in-frame SfiI or NotI restriction site to facilitate the cloning of the PCR product into the phagemid pCANTAB5E vector.
pCANTAB5E was designed so that the antibody V region could be cloned between the leader sequence and the main body of the M13 gene III. pCANTAB 5E also contains a sequence encoding a peptide E tag followed by an amber translation stop codon at the junction between the cloned scFv and the gene III sequence. The ligation mixture was transformed into a suppressor-deficient E. coli strain (HB2151). The expression of the scFv was induced by adding isopropyl beta-D-thiogalactoside (IPTG) to a final concentration of 1 mM for 3 h. The cells were collected by centrifugation at 5,000 g for 20 min and incubated in 1 mM Ethylene diamine tetraa cetic acid in PBS for 10 min on ice to obtain the periplasmic fraction. The periplasmic extract was filtered through a 0.45-μm filter, and the scFv was purified by passage over an anti-E tag affinity column equilibrated with PBS. After the column was washed with 0.1 M glycine-HCl (pH 5.0), the bound scFvs were eluted with 0.1 M glycine-HCl (pH 2.8). Each eluted 1.5 ml fraction was treated with 175 μl of 2.0 M Tris-HCl (pH 8.0) to neutralize the pH.
Next step of the methodology is cloning of the variable fragment of the C3A8 monoclonal antibody gene. A cool start protocol was used to amplify the heavy and light chain variable regions. To the entire first strand reaction mixture (33 μl), 40 pmol of each primer ( Primers B and F for amplification of light variable chain (VL) for amplification of VL or HB and HF for amplification of VH) and 2.5 μl Taq polymerase (Promega) was added, with a final reaction volume of 100 μl. The reactions were incubated on ice before running the following PCR program: denaturation at 92°C for 5 min followed by seven cycles of 1 min at 92°C, 30 s at 63°C, 50 s at 58°C, and 1 min at 72°C, and 23 cycles of 1 min at 92°C, 30 s at 63°C, and 1 min at 72°C. The reaction products were analyzed on a 1% agarose gel.
Moreover, PCR amplification of the scFv gene, the V L and V H PCR products were purified with a QIAquick PCR Purification Kit (Qiagen). Approximately, 30 ng of each V L and V H fragment was combined using splicing by overlap extension PCR (31). Taq polymerase was added, and the solution was kept on ice. The following program was used for amplification: denaturation at 95°C for 5 min followed by five cycles of 1 min at 95°C, 30 s at 63°C, 50 s at 58°C, and 3 min at 72°C without primers. After the addition of the outer primers, the reaction was denatured at 95°C for 5 min, after which the following programs were run: Five cycles of 1 min at 95°C, 30 s at 63°C, 50 s at 58°C, and 3 min at 72°C and 35 cycles of 1 min at 95°C, 1 min at 55°C, and 3 min at 72°C, with a final extension at 72°C for 7 min. Following amplification, the PCR mixture was analyzed via 1% agarose gel electrophoresis.
In the Isolation of immunoglobulin sequences from the C3A8 hybridoma, the C3A8 hybridoma antibody sequences were cloned and assembled. To obtain enough scFv products, five assembly PCRs were performed as described above, and the resulting products were pooled, precipitated with ethanol, and washed. The pooled DNA was purified on a 1% gel, and approximately 1 μg of the gel-purified scFv fragment was digested in an 85-μl reaction at 50°C for 4 h. The digested PCR insert (750 bp) was purified in a 1% agarose gel, and the concentration was determined by running an aliquot of the purified DNA in a 1%-gel and comparing the intensity with the known amounts of DNA in the ladder. The phage display vector pCANTABE5 was prepared using a Wizard Miniprep Kit (Promega), as recommended by the manufacturer. pCANTABE5 (10 μg) was digested with 2 μl of SfiI in a 100-μl reaction volume at 50°C for 4 h and purified in a 0.5% gel as described above. After digestion, 200 ng of the vector was ligated with 20 ng of scFv (a molar vector to insert ratio of 1:1.5) and transformed into E. coli strain TG1 (Stratagene). For the isolation of the light and heavy chain sequences, the transformants were spread on five non-expression plates (Nalidixic acid (NE); 2 × yeast-tryptone [YT] containing 1% glucose and 25 μg/ml chloramphenicol), which were incubated overnight at room temperature. All of the resulting colonies were transferred into 5 ml of NE, and the cultures were stored at −80°C after the addition of 15% glycerol.
Recombinant phage was rescued using the following steps: The titers of the phage stocks were determined by plating. The phage culture (50 μl) was inoculated into 25 ml of NE medium and grown at 37°C with shaking. When an optical density at 600 nm (OD600) between 0.5 and 1.0 was reached, 500 μl of M13K07 (Pharmacia Biotech) helper phage (approximately 10 12 PFU), 37.5 μl of 1 M IPTG (final IPTG concentration, 0.5 mM), and 50 ml of NE were added, and the culture was shaken overnight at 26°C and 225 rpm for phage production.
Two hours after IPTG induction, kanamycin was added to the culture to a final concentration of 30 μg/ml. The next day, the culture medium was collected and centrifuged at 4,000 g for 10 min at 4°C to eliminate bacterial cells. To precipitate the phage particles, 20 ml of 5 times the molecular weight of polyethylene glycol (PEG)-NaCl (20% PEG 8000, 2.5 M NaCl) was added to the supernatant (approximately 70 ml), which was then incubated on ice for 30 min. The phage particles were then precipitated via centrifugation (10,000 g at 4°C for 15 min). The supernatant was decanted, and the pellet was resuspended in 30 ml of phosphate-buffered saline (PBS). The phage solution was mixed with 5.5 ml of PEG-NaCl on ice for 20 min and centrifuged again at 10,000 g at 4°C for 15 min. The supernatant was discarded, and the phage pellet was resuspended in 2 ml of PBS containing 10% glycerol for storage at 4°C. The phage solution was either subjected to further panning after titers were determined or was used in a phage ELISA to determine the antibody activity. Only freshly prepared phage was used for panning to optimize the binding ability.
Nunc immunotubes were coated overnight with 1 ml of MCF-7 whole cells in PBS 1 × 10 6 of MCF-7 cells in 10 ml of PBS, pH 7.4) at 4°C. The next day, the tubes were washed three times with PBS. For blocking, the tubes were filled with 2% bovine serum albumin (BSA) in PBS and incubated at 37°C for 2 h. The tubes were washed three times with PBS, and 10 12 PFUs of phage in 1 ml of 2% BSA in PBS was added. The tubes were left in a stationary position at 37°C for 1 h, and the unbound phage in the supernatant was discarded. The tubes were washed 10 times with PBS containing 0.1% Tween 20, and then the tubes were washed 10 times with PBS to remove the detergent. The excess PBS was discarded, and the phage was eluted by adding 1 ml of 100 mM triethylamine (700 μl of triethylamine in 50 ml of water, diluted on the day of use), with the tube kept stationary at room temperature for 10 min during the incubation as described before. 
The eluted 1 ml of phage was added to another micro-centrifuge tube containing 0.5 ml of 1 M Tris (pH 7.4) for quick neutralization. Half of this volume (750 μl) was added to 5 ml of mid-log phase (OD600 of 0.6) cultures of E. coli HB2151. The cells were shaken at 250 rpm at 37°C for 1 h and then centrifuged at 3,000 g for 5 min. The supernatant was carefully discarded, and the cells were resuspended in 500 μl of 2× (YT) Bacto Tryptone. The entire contents were spread onto five NE plates (100 μl per plate) and grown overnight at 26°C. After overnight growth, 1.5 ml of 2× (YT) Bacto tryptone medium was added to each plate, and the colonies were scraped off the plates and pooled into micro-centrifuge tubes. If a further round of selection was required, 25 ml of NE medium was inoculated with 50 μl of these cells and grown to an OD600 of 1.0. The phagemid rescue was performed as described above. The remaining harvested cells were stored at −70°C.
In screening for binding via ELISA, 48 colonies were picked after the second panning for the binding test against MCF-7 cells. MCF-7 cells were harvested via centrifugation and stored in glycerol buffer at 20°C. ELISA plates were coated with 1 × 10 6 of MCF-7 in (100 μl/well) in PBS at 4°C and incubated overnight. The wells were then rinsed 3 times with PBS and blocked with 300 μl of 2% BSA in PBS per well for 2 h at 37°C. For the preparation of the phage antibody supernatant to be used as the first antibody in the ELISA, HB2151 colonies were grown separately in 2 ml of NE at 37°C until an OD600 of 0.5 was reached. IPTG (1 mM) and M13K07 helper phage (109 PFUs) were added, and the culture was grown overnight at 26°C in a shaking incubator at 225 rpm.
The phage supernatants were collected via centrifugation at 3,000 g at 4°C for 10 min, and an equal volume of 2% BSA in PBS (MPBS) was added to the phage supernatants, which were then used directly in the assay. After blocking with 5% MPBS, the wells were rinsed three times with PBS and loaded with 100 μl of the phage supernatant prepared as described above. The plate was incubated for 1.5 h at room temperature, and the wells were washed three times for 2 min each with 0.1% Tween 20 in PBS. An HRP-conjugated anti-M13 antibody (100 μl of 1:1,000 dilution in 2% BSA in PBS, Amersham Pharmacia Biotech) was added, and the plates were incubated at room temperature for 1 h. The plates were washed as described above. The substrate was prepared by mixing 20 ml of citric acid buffer, 8 mg of O-phenylenediamine, and 80 μl of 3% H 2 O 2 , and 150 μl of the substrate was added to each well. The plates were incubated in the dark for at least 10 min at room temperature, and the reaction was stopped by adding 50 μl of 1 M H 2 SO 4 per well. The plates were read at 492 nm in a 312 ELISA Auto-Reader (Biotek-340).
The C3A8 scFv gene was DNA-sequenced with an Applied Bio-systems model 377 automated DNA Sequencing System (FIRST BASE Laboratories) with the pCANTAB5E Sequencing Primer Set primers. Single-strand DNA (ssM13 DNA) was prepared using a QIAprep M13 Purification Kit, as recommended by the manufacturer, whereas double-strand DNA was prepared using a PureLink Quick Plasmid Miniprep, as recommended by the manufacturer.
Soluble expression of single-chain antibodies was produced using the supernatant from antigen-binding recombinant phages (2 μl) was added to log phase E. coli (strain HB2151) in 2 times (YT) Bacto-tryptone medium (400 μl), and the cultures were incubated at 37°C for 30 min with intermittent gentle shaking. At the end of the incubation, the culture was streaked onto a Bacto-tryptone, Bacto yeast , and ,NaCl containing Nalidixic acid (SOBAG-N) plate (SOBAG containing 100 μg/ml Nadixic acid), and the plates were incubated at 30°C overnight. A single infected colony on SOBAG-N plate was picked and cultured in 2× (YT) Bacto-tryptone -AG medium (5 ml) containing 100 μg/ml ampicillin and 2% glucose overnight at 30°C. The overnight culture was diluted 1:10 with fresh 2(YT) Bacto-tryptone medium -AG medium and incubated at 30°C for 1 h. After the incubation, the culture was centrifuged at 1500 g for 20 min, and the supernatant was discarded. The pellet was then resuspended in 2 × Bacto tryptone-AI medium (2 YT) medium containing 100 μg/ml ampicillin and 1 mM IPTG), and the cell suspension was incubated for additional 5 h. At the end of the incubation, the culture was divided into two separate 50-ml centrifuge tubes and centrifuged as detailed above. To isolate periplasmic scFvs, one of the resulting pellets was resuspended in 0.5 ml of ice-cold 1× Tris -HCl EDTA Buffer Saline (TES) buffer. Dilute TES buffer (0.75 ml of ice-cold 0.2 × TES buffer) was added, and the mixture was vortexed and incubated on ice for 1 hour. Finally, the supernatant containing periplasmic scFv was collected via centrifugation at full speed for 10 min. For the preparation of whole-cell extracts, the second pellet was resuspended in 0.5 ml of PBS and boiled for 5 min. The extract was centrifuged as detailed above. If not used immediately, the isolated scFvs were stored at 20°C prior to use in the immuno-detection experiments.
SDS-PAGE and Western blot was used to analyze scFv protein expression. Therefore, the sample was loaded into SDS polyacrylamide gels and run at 100 V for 1.5 h. The gel was stained with Coomassie blue (0.1% Coomassie blue, 40% methanol, and 10% glacial acetic acid) and destained with buffer (10% methanol and 10% glacial acetic acid) overnight. The protein was transferred to a PVDF membrane (Immobilon-P, Millipore, USA) for western blot analysis (Appendix C) using a semi-dry electro-blotting system (Bio-Rad, USA) for 45 min at 15 V in transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol). The membrane was blocked with blocking buffer (1% BSA in PBS) for 1 h and washed in washing buffer (0.05% Tween 20 in PBS). The membrane was then incubated with an HRP-conjugated anti-E tag antibody (diluted 1:1,000, Amersham Pharmacia Biotech) at room temperature for 30 min. The membrane was washed three times in washing buffer for 5 min each. The detection reagent was prepared in blocking buffer containing 100 mM Tris and 150 mM NaCl. The membrane was washed, and the 4CN (4-chloro-1-napthol substrate, Sigma, USA) substrate was added to the membrane. After 5 min, the reaction was stopped by the addition of distilled water. The membrane was examined for a protein band of the correct size (32-kDa) and visualized on a Kodak film.
Finally, the indirect ELISA used to characterize the scFv antibody binding activity against MCF-7 was modified as follows. MCF-7 cells were plated in ELISA plates (100 μl/well in RPMI medium) and incubated overnight at 37°C with 5% CO2. The wells were rinsed three times with PBS and blocked with 2% BSA for 2 h at 37°C.The wells were rinsed three times with PBS and blocked with 2% BSA for 2 h at 37°C. The wells were then rinsed three times with PBST, and 100 μl of phage supernatant or periplasmic extract generated as described above was added. The plates were incubated at room temperature for 1 h, and then washed three times with PBST. An HRP-conjugated anti-E tag antibody (100 μl of a 1:2,000 dilution in blocking buffer; Amersham Pharmacia Biotech) was added, and the plates were incubated at room temperature for 1 h. The plates were then washed, and 100 μl of substrate was added per well. The plates were incubated for at least 50 min at room temperature in the dark and then read at 405 nm in an ELISA Auto-Reader (Bio-Tek Instruments, Winooski, VT).
An HRP-conjugated anti-E tag antibody (Amersham Pharmacia Biotech) was used in the ELISA. The conjugate was diluted 1:100, 1:200, 1:500, 1:1000 1:2000, and 1:4000 in washing buffer, and the optimum incubation time was then determined. The same amount of the scFv antibodies was tested at different serial concentrations. The scFv was diluted 1:2, 1:4, 1:8, 1:16, and 1:32. The scFv concentration at the 1:32 dilution was equivalent to 15 ng.
The binding activity of scFv protein to MCF-7 cells was also analysed using a Flow Cytometry test, the flow cytometry Fluorescence-activated cell sorting (FACS) Calibur and Cell Quest software was used (Becton Dickinson, Mountain View, CA). In this test, the cells were stained with scFv protein to monitor binding of scFv fusion protein. The numbers of 5 × 10 5 cells were incubated for 24 h and 48 h on ice with 2 ml of scFv at a concentration of 20 μg/ml. Non-stained MCF-7 cells served as a control for background staining. The cells were washed with PBA buffer and then incubated with HRP-conjugated anti-E tag antibody monoclonal antibody in PBS buffer (1:500) at room temperatures for 1 h (novagen, Bad Soden, Germany). Cells were washed and incubated with fluorescein-iso-thiocyanate (FITC)-conjugated goat anti-mouse IgG (novagen, Bad Soden, Germany) in PBS buffer (1:100) for 1 h. After a final wash, the cells were analysed by FACS.
| > Results|| |
In order to prepare the scFv DNA fragment, the V H and V L cDNAs were amplified using PCR. The V H and V L amplification products were visualized on agarose gels as seen in [Figure 1] a, and were approximately 340 bp (lane 1) and 320 bp (lane 2) in length respectively. The purified V H and V L cDNAs were assembled into a single gene using a DNA linker fragment, and a PCR-amplified scFv DNA fragment (approximately 750 bp) was obtained [Figure 1]b and c. The 750-bp cDNA band was further purified with a Gene-clean II Kit. Following digestion with the restriction enzymes SfiI and NotI, the scFv DNA fragment was ligated to the phagemid vector, pCANTAB5E. Following transformation, the E. coli TG1 cells contained pCANTAB5E plasmids with an approximately 750-bp insert.
|Figure 1: (a) Amplification of the variable heavy and variable light chain genes from the hybridoma line C3A8. The amplified VH (lane 1) and VL (lane 2) products were electrophoresed in a 1.2% agarose gel in 0.5 TAE and stained with ethidium bromide. Lane M 100 bp DNA ladder (New England Biolabs, UK) was used for size indication. The amplified VH and VL DNAs were about 340 bp and 320 bp respectively; (b) The VH and VL were then assembled into a single-chain fragment through the following assembly PCR. Amplification of the assembled single-chain fragment variable (scFv) produces. The amplified scFv products (lane 1and 2) were electrophoresed in a 1.0% agarose gel in 0.5 TAE and stained with ethidium bromide. The amplified ScFv DNAs was about 750 bp of scFv and unconnected VH and VL as secondary product in each lane; (c) Gel purification products shown in lanes 1 and 2 were just purified 750 bp band of scFv. The scFv product was further purified and cloned into the phagemid vector (pCANTAB 5E) to construct the single-chain antibody phage display cassette|
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The phage-expressed antibodies were subjected to biopanning to selectively capture recombinant phages expressing scFvs that recognize MCF-7 cells line bound to an immuno-tube. Two panning cycles were carried out, and the phage antibody pools prepared after no, one, or two rounds of panning were tested for MCF-7cells line specific binding in a phage ELISA. As seen in [Figure 2], the ELISA signal increased in the final round of panning. Prior to panning, the phage antibody gave a very weak signal against MCF-7 cells line, with an OD405 of only 0.372. However, the OD405 increased with the number of rounds of panning performed. The bacteriophages containing nucleotide sequences encoding a functional scFv sequence were enriched by repeating the biopanning. After two rounds of panning, four antigen-positive phage clones were selected from 45 preselected phage clones using ELISA. The four clones (D11, B7, B1, and A10) showed the strongest signal [Table 1]. Only one of the four clones (clone B7) was selected for further study [Figure 3].
|Figure 2: Binding specificity of phage from individual clones in ELISA test. Phage was produced from individual colonies isolated after two rounds of panning or from the unpanned library (R0). 109 colony-forming units were added to each well of a micro titer plate that had been pre-coated with MCF-7 cells line and bound phage detected with anti-M13 antibody|
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|Figure 3: Gel electrophoresis screening of the highest ELISA absorbents has been showed. The gel electrophoresis showed that B7 single colony gave the best sequences identity and has the brighter band among the rest of the colonies|
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The DNA sequencing was used to identify the gene families for the V L and V H regions, nucleotide sequencing was carried out. As shown in [Figure 4], the deduced amino acid sequence of the anti-MCF-7 scFv confirmed the expected protein structure in which the V L and V H regions were connected with three consecutive GGGGS repeats, which resulted from the linker sequence [Figure 1b and c]. A comparison of the nucleotide sequence with the sequences deposited in the NCBI database revealed that the V H and V L regions were highly homologous to the murine VH1 and VL1 the mouse families respectively. Interestingly, a homology search of the GenBank database indicated that the scFv resembled the antigen-recognition site of the single chain antibody against rice stripe virus protein P20 [synthetic construct], with 99% homology for V H and 100% homology for V L (data not shown).
|Figure 4: PCR amplification for single stranded DNA produced from soluble phage induced in media after large scale production of scFv-phage fusion. Seven colonies were randomly chosen for PCR detection using R1 and R2 primers (Pharmacia, Sweeden). The PCR products were electrophoresed in a 1% agarose gel in 0.5 TAE and stained with ethidium bromide. Lane M: 100 bp DNA ladder. Lane 1-2 double `stranded DNA as a 1000 bp. Lane 3, 5, 6 and 7 single stranded DNA detected as a 1000 bp. Lane 4: Negative control of bacterial colony carrying pCANTAB 5E vector only was used for the PCR detection|
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|Table 1: Screening for phage positive clones displaying antigen binding antibodies against MCF7 cells before production of soluble antibodies in Escherichia coli TG1|
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In order to produce a soluble form of scFv protein, the cell Lysates were generated from the Escherichia coli HB2151 cells transformants with scFv DNA insert, and the immunological properties were examined via Western blotting against MCF-7 cells lysates; Escherichia coli HB2151 cells transformed with pCANTABE5 were used as a negative control [Figure 5]a, lane 1]. To purify the recombinant scFv, the location of the protein in the transformants was examined. As seen in [Figure 5]a, the scFv was found in both the periplasmic and whole-cell lysates. Subsequently, the large-scale production of the anti-MCF-7 scFv was performed using the periplasmic fraction. The periplasmic fraction generated from a 1 L culture was passed over an anti-E tag affinity column, and a 32-kDa protein was purified [Figure 5]a, lanes 2 and 3].
|Figure 5: (a) SDS-PAGE for detection of soluble C3A8 ScFv from crude cells extract, periplasmic and supernatant extracts. Lane 1: PCANTAB 5E phagemid vector without insert (blank). Lane 2: Periplasmic extracts. Lane3: Supernatant extracts Lane 4: Crude cells extract (b) SDS-PAGE for detection of purified scFv antibody, lane 2 and 3 from preiplasmic as 32 KDa band was appeared in size. Lane1 the expressed pCANTAB 5E without insert as blank (c) Western blot of scFv antibody detected with an anti-E tag antibody. Inserts were grown in the expression vector pCANTAB 5E. Denatured protein was separated by SDS-PAGE then transferred onto PVDF membrane. The membrane was probed using Anti-E tag antibody and finally developed using 4-CN substrate. A 32 kDa band was visualized on the membrane Lane 1: detection of soluble C3A8 ScFv from periplasmic. Lanes 2: vector without insert (blank). Lane M: PageRuler™ Prestained Protein Ladder, (Fermentas)|
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Western blot analysis was carried out. Solubilized vesicular proteins were transferred onto a PVDF membrane, and the membrane was probed with scFv antibody from preiplasmic space. As seen in [Figure 5]b (lanes 1 and 2), a single 32-kDa protein was seen in the periplasmic extracts from cells transformed with the anti-MCF-7 scFv construct when the membranes were probed with the E tag-specific antibody [Figure 5]c,[ lanes 2, 3, and 4]. The scFv was not observed in the non-induced cultures or in the cells transformed with the empty vectors [Figure 5]c, [lane 1]. As shown in [Figure 5]c the indirect ELISA compared the binding activity of constructed scFv antibody with the parents monoclonal antibody isolated from Hybridoma C3A8.
After the expression of soluble scFv in Escherichia coli HB2151, the supernatants and pellets were checked for the presence and reactivity of the scFv via ELISA and Western blot. Based on the ELISA results, the scFv were secreted into the supernatant and present in the periplasmic extract reacted with MCF-7 cells [Figure 6]a. The purified soluble antibodies were also used in an indirect ELISA. The scFvs retained the ability to capture measurable antigen from less than 1 × 10 6 cells MCF-7/ml. Under these conditions, all of the scFvs from different clones gave values that were substantially higher than values obtained using the C3A8 monoclonal antibody [Figure 6]b. Importantly, only low non-specific activity was observed with the blank vector. The comparative affinities of the parental C3A8 hybridoma and the best representative scFv were evaluated using the initial ELISA data.
In addition to these results, flow cytometry test shows the binding reactivity of the best representative scFv fragment expressed in preiplasmic space with MCF-7 cell line [Figure 7]a and c. Also, non-bound MCF-7 cell line proved the specific binding of MCF-7 cell line [Figure 7]b. Thus, the expressed scFv protein in preiplasmic space did detectably improve the binding specificity of the antigen-combining sites.
|Figure 6: (a) In indirect ELISA graph, the reactivity of scFv antibody and C3A8 monoclonal antibody recognizing MCF-7 breast cancer cells. The green color reaction obsereved showed the degree of affinity in both types of antibodies. pCANTAB 5E vector without insert was work as blank in this test. In this fig the scFv antibody and C3A8 monoclonal antibody concentrations were serially diluted, while anti E-tag antibody conjugated to horseradish peroxidase (HRP) added to the reaction were constant; (b) in indirect ELISA plat, the reactivity of scFv antibody and C3A8 monoclonal antibody recognizing MCF-7 breast cancer cells. The green color reaction observed showed the degree of affinity in both types of antibodies. pCANTAB 5E vector without insert was work as blank in this test. In this fig the scFv antibody and C3A8 monoclonal antibody concentrations were serially diluted, while anti E-tag antibody conjugated to HRP added to the reaction were constant|
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|Figure 7: (a) Forty-eight hour of MCF-7 cells stained with purified ScFv antibody, incubated 48 h, 79% of cells stain positive for annexin V; (b) Negative control In this test, 10.2% of MCF-7 cells were positive for annexin V, an early marker of apoptosis; (c) MCF-7 cells stained with purified scFv antibody, incubated 24 h, 38% of cells stain positive for annexin V|
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| > Discussion|| |
Immunological methods are widely used as they are rapid and relatively economical. In the last few years, a variety of molecular approaches to develop antibodies have become widely available. , Generating polyclonal antibodies is time consuming, requires large animals, and may be subject to the vagaries of biological variability. Furthermore, hybridoma generation is slow, expensive, and effort-intensive, and many rounds of testing may be required to isolate a satisfactory antibody.  However, the use of phage display in combination with antibody gene cloning can minimize these problems. Recombinant antibody cloning coupled with Recombinant antibody cloning from hybridoma cells is a relatively well-established assay that has been used for the development of better diagnostic reagents.  Several years after the initial description of hybridomas, phage display was introduced.  In contrast to the conventional hybridoma technology initially described for the production of monoclonal antibodies,  phage display is not based on immortalization and cloning of the antibody-producing lymphocytes. Rather, the screening of antibody libraries can recapitulate the vertebrate immune system in vitro.
In this study, the techniques and phage display system of Krebber et al.  was used with C3A8 hybridoma cells, as they provided a better representation of the antibody gene repertoire compared with spleen cells.  The extended primer set can incorporate all of the mouse V H and V L sequences in the Kabat database  and can be modified for other species as well. In addition, the system included an E tag at the N terminus of the light chain, allowing for easy purification using commercial kits.
Having enough of the light and heavy chain cDNAs was critical to the success of the assembly PCR. To obtain enough light and heavy chain cDNA, the entire first-strand reaction was used for amplification. The amplified V H and V L cDNAs were approximately 340 bp and 320 bp in length respectively [Figure 1]a. Additionally, the concentration of both cDNAs had to be determined to keep their amounts the same, which was critical for the success of the subsequent assembly PCR. The most accurate measure was obtained using gel electrophoresis, and the intensity of bands was compared with the known quantities in the DNA ladder. These amounts were assembled in the subsequent assembly PCR, which ultimately produced the 750-bp scFv DNA [Figure 1]b. More than one assembly PCR was performed to obtain enough pure scFv DNA, as shown in [Figure 1]c.
The amplified V H and V L genes were joined with a linker and expressed as a single polypeptide chain. The joining of the V H and V L regions with peptide linker enables the intra-molecular association of the variable regions, allowing for the generation of variable domain combinations that react weakly.  The linkers that connect the various domains of multi-domain proteins are stretches of amino acids that establish communication between the different domains and functional modules.  Therefore, the flexible 15-amino acid peptide linker used in this study (GGGGSGGGGSGGGGS) was specifically designed to bridge the 3.5-nm gap between the C terminus of the VH chain and the N terminus of the VL chain. , This construction facilitates chain pairing and minimizes the refolding and aggregation problems encountered when the two chains are expressed individually. ,, Importantly, the affinity and stability of scFvs with the (GGGGS) 3 linker are similar to those of the native antibody, even though the scFvs are more specific. 
As described in many studies, two rounds of panning were sufficient to enrich for the desired sequences. Recombinant libraries of human antibody genes that are selected via phage display and biopanning have provided a convenient tool for the development of gene therapies and diagnostic tools. ,,, As the results of the indirect ELISA did improve with additional biopanning [Figure 2], the scFvs expressed on the surface of the phage were highly specific for MCF-7 cells.
As seen in [Figure 3], in non-suppressor Escherichia coli strains, such as HB2151, the stop codon is recognized, and protein synthesis terminated at the end of the scFv gene. In addition, the pCANTAB5E used in this study contains a group 3p leader sequence that directs the transport of the scFv to the periplasm. Additionally, while a small amount of ssDNA was purified in the first cultures, to obtain enough ssDNA for PCR amplification and sequencing, the second isolation required a 28-h culture [Figure 4]. The soluble scFv accumulated in the periplasm in a small amount, necessitating large-scale cultures with extended incubation times to produce large amounts of the antibody. , The soluble scFv was observed as a 32-kDa band via SDS-PAGE and Western blot [Figure 5]a, and sufficient material could be collected from the medium. Based on the indirect ELISA data [Figure 6]a, the performance of the scFv was superior to that of the parental monoclonal antibody [Figure 6]b. While the ELISA signal from clone B7 was slightly lower than that from the C3A8 monoclonal antibody, the binding of the scFv to MCF-7 cells was highly specific, as evidenced by the poor binding of the negative control. Furthermore, extracts from cells transformed with the empty pCANTAB5E vector did not bind to MCF-7 cells [Figure 6]a and b. Moreover, the recombinant scFv showed significant reactivity toward the mcf7 cell of approximately 79% by scFv as measured by flow cytometry analysis as shown in [Figure 7]a and b, but not to non-stained MCF-7 cell line with scFv protein as shown in [Figure 7]c. An additional control was performed to exclude that any recombinant scFv protein may have cross reactivity because of antigens common design.
In conclusion, we report the construction and expression of a functional scFv recognizing the MCF-7 breast cancer cell line, and soluble scFvs specific for MCF-7 cells are available. This reagent will serve as a promising targeting vehicle for both in vitro and therapeutic applications targeting MCF-7 breast carcinoma cells and also provides a stable genetic source for the scFv antibody. Furthermore, the expression of the scFv in the periplasm of HB2151 cells was characterized using indirect ELISA and compared with the parental monoclonal antibody. However, because the scFv is smaller than the parental monoclonal antibody, the affinity of the scFv antibody was slightly lower. Moreover, MCF-7 breast carcinoma cells showed binding reactivity toward scFv protein expressed in preiplasmic space in flow cytometry test when tested using an annexin V-based apoptosis assay. The binding intensity gradually increased, with 79% of the cells displaying scFv protein binding after 48 h.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]