The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana

 

Show simple item record

dc.contributor.advisor Meyers, Ann Elizabeth
dc.contributor.advisor Meggersee Rosemary
dc.contributor.advisor Rybicki, Ed
dc.contributor.author Ndlovu, Siphumelele
dc.date.accessioned 2020-10-14T11:50:46Z
dc.date.available 2020-10-14T11:50:46Z
dc.date.issued 2020_
dc.identifier.citation Ndlovu, S. 2020. The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana. . ,Faculty of Science ,Department of Molecular and Cell Biology. http://hdl.handle.net/11427/32303 en_ZA
dc.identifier.uri http://hdl.handle.net/11427/32303
dc.description.abstract Monoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents.
dc.subject Molecular and Cell Biology
dc.title The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana
dc.type Master Thesis
dc.date.updated 2020-10-14T11:50:21Z
dc.language.rfc3066 eng
dc.publisher.faculty Faculty of Science
dc.publisher.department Department of Molecular and Cell Biology
dc.type.qualificationlevel Masters
dc.type.qualificationlevel MSc
dc.identifier.apacitation Ndlovu, S. (2020). <i>The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana</i>. (). ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/32303 en_ZA
dc.identifier.chicagocitation Ndlovu, Siphumelele. <i>"The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana."</i> ., ,Faculty of Science ,Department of Molecular and Cell Biology, 2020. http://hdl.handle.net/11427/32303 en_ZA
dc.identifier.vancouvercitation Ndlovu S. The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana. []. ,Faculty of Science ,Department of Molecular and Cell Biology, 2020 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/32303 en_ZA
dc.identifier.ris TY - Master Thesis AU - Ndlovu, Siphumelele AB - Monoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents. DA - 2020_ DB - OpenUCT DP - University of Cape Town KW - Molecular and Cell Biology LK - https://open.uct.ac.za PY - 2020 T1 - The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana TI - The isolation of single chain variable region fragments (scFvs) from a phage display library, and expression of the isolated scFvs in Nicotiana benthamiana UR - http://hdl.handle.net/11427/32303 ER - en_ZA


Files in this item

This item appears in the following Collection(s)

Show simple item record