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The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli

Toxoplasmosis is a disease caused by infection of a parasitic protozoan known as Toxoplasma gondii, which has economic implications due to neonatal loss and abortion in livestock animals. In human adults, it is usually asymptomatic but can cause complications in immunocompromised persons and pregnan...

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Main Author: Fatin Iffah Rasyiqah , Mohamad Zoolkefli
Format: Thesis
Published: 2018
Subjects:
Q Science (General)
QH301 Biology
Online Access:http://studentsrepo.um.edu.my/12033/1/Fatin_Iffah.pdf
http://studentsrepo.um.edu.my/12033/2/Fatin_Iffah_Rasyiqah.pdf
http://studentsrepo.um.edu.my/12033/
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spelling my.um.stud.120332021-02-24T19:45:10Z The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli Fatin Iffah Rasyiqah , Mohamad Zoolkefli Q Science (General) QH301 Biology Toxoplasmosis is a disease caused by infection of a parasitic protozoan known as Toxoplasma gondii, which has economic implications due to neonatal loss and abortion in livestock animals. In human adults, it is usually asymptomatic but can cause complications in immunocompromised persons and pregnant women which leads to congenital effects on newborn babies. As a therapeutic approach to combat the disease, biopharmaceutical products, such as recombinant antibody targeting to T. gondii tachyzoite, has been introduced as an alternative to drugs and vaccines. Innovative approaches for the expression and production of these novel biopharmaceutical products which increased complexity towards disease targets have been the subject of recent investigations. Plants have now emerged as a preferred host to express and produce these recombinant proteins as it provides advantages in term of efficiency, cost, scalability, safety and compatibility as compared to prokaryotes and mammalian cell lines. Thus, in this study, the target antibody, an anti-Toxoplasma scFv encoded by the TP60 gene was transformed into leaf explants of Nicotiana tabacum cv. SR1 using Agrobacterium tumefaciens strain LBA4404. The constructs were transformed in a series of experiments in tandem with different enhancer elements comprising proteinase inhibitors or a KDEL retention sequence or combinations of both elements in order to assess their ability to stabilize and enhance the scFv production. Two different proteinase inhibitors that have been used in this study were Oryzacystatin inhibitor (OCPI) (a cysteine inhibitor) and Bowman-Birk inhibitor (BBI) (a trypsin and chymotrypsin inhibitors). β-glucuronidase (GUS) and green fluorescence protein (GFP) assays as well as PCR results confirmed the presence of transgenes in the tobacco genome that was stably inherited into T1 generation. For Western blot analysis, the selected lines of transgenic tobacco plants transformed with pTP60 construct in combination with BBI-KDEL (pTP60BBIKDEL) elements was found to produce the highest anti-Toxoplasma scFv recombinant antibody accumulation at the expected size of ~ 54 kDa (homodimer formation) which was at first observed at ~ 27 kDa (monomer) in T0 tobacco plants. No expression was detected in non-transformed plants. The results were also confirmed at the mRNA level using real-time PCR analysis with the BBI-KDEL tandem constructs expressing TP60 gene approximately 19-fold higher as compared to plants transformed with the pTP60 construct in the absence of any proteinase inhibitor and/or KDEL. Interestingly, the introduction of all of these elements did not or only negligibly affected the growth and development of the transgenic tobacco plants as compared to the wild-type. Our results confirmed the ability of plants to function as bio-factories for recombinant protein production and showed that the use of proteinase inhibitor and KDEL elements may be useful in improving the yield of the targeted antibody proteins. 2018-07 Thesis NonPeerReviewed application/pdf http://studentsrepo.um.edu.my/12033/1/Fatin_Iffah.pdf application/pdf http://studentsrepo.um.edu.my/12033/2/Fatin_Iffah_Rasyiqah.pdf Fatin Iffah Rasyiqah , Mohamad Zoolkefli (2018) The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli. Masters thesis, University of Malaya. http://studentsrepo.um.edu.my/12033/
institution Universiti Malaya
building UM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaya
content_source UM Student Repository
url_provider http://studentsrepo.um.edu.my/
topic Q Science (General)
QH301 Biology
spellingShingle Q Science (General)
QH301 Biology
Fatin Iffah Rasyiqah , Mohamad Zoolkefli
The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli
description Toxoplasmosis is a disease caused by infection of a parasitic protozoan known as Toxoplasma gondii, which has economic implications due to neonatal loss and abortion in livestock animals. In human adults, it is usually asymptomatic but can cause complications in immunocompromised persons and pregnant women which leads to congenital effects on newborn babies. As a therapeutic approach to combat the disease, biopharmaceutical products, such as recombinant antibody targeting to T. gondii tachyzoite, has been introduced as an alternative to drugs and vaccines. Innovative approaches for the expression and production of these novel biopharmaceutical products which increased complexity towards disease targets have been the subject of recent investigations. Plants have now emerged as a preferred host to express and produce these recombinant proteins as it provides advantages in term of efficiency, cost, scalability, safety and compatibility as compared to prokaryotes and mammalian cell lines. Thus, in this study, the target antibody, an anti-Toxoplasma scFv encoded by the TP60 gene was transformed into leaf explants of Nicotiana tabacum cv. SR1 using Agrobacterium tumefaciens strain LBA4404. The constructs were transformed in a series of experiments in tandem with different enhancer elements comprising proteinase inhibitors or a KDEL retention sequence or combinations of both elements in order to assess their ability to stabilize and enhance the scFv production. Two different proteinase inhibitors that have been used in this study were Oryzacystatin inhibitor (OCPI) (a cysteine inhibitor) and Bowman-Birk inhibitor (BBI) (a trypsin and chymotrypsin inhibitors). β-glucuronidase (GUS) and green fluorescence protein (GFP) assays as well as PCR results confirmed the presence of transgenes in the tobacco genome that was stably inherited into T1 generation. For Western blot analysis, the selected lines of transgenic tobacco plants transformed with pTP60 construct in combination with BBI-KDEL (pTP60BBIKDEL) elements was found to produce the highest anti-Toxoplasma scFv recombinant antibody accumulation at the expected size of ~ 54 kDa (homodimer formation) which was at first observed at ~ 27 kDa (monomer) in T0 tobacco plants. No expression was detected in non-transformed plants. The results were also confirmed at the mRNA level using real-time PCR analysis with the BBI-KDEL tandem constructs expressing TP60 gene approximately 19-fold higher as compared to plants transformed with the pTP60 construct in the absence of any proteinase inhibitor and/or KDEL. Interestingly, the introduction of all of these elements did not or only negligibly affected the growth and development of the transgenic tobacco plants as compared to the wild-type. Our results confirmed the ability of plants to function as bio-factories for recombinant protein production and showed that the use of proteinase inhibitor and KDEL elements may be useful in improving the yield of the targeted antibody proteins.
format Thesis
author Fatin Iffah Rasyiqah , Mohamad Zoolkefli
author_facet Fatin Iffah Rasyiqah , Mohamad Zoolkefli
author_sort Fatin Iffah Rasyiqah , Mohamad Zoolkefli
title The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli
title_short The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli
title_full The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli
title_fullStr The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli
title_full_unstemmed The effect of KDEL and PPI genes co-expression on the anti-toxoplasma scFV activity in TRANSGENIC Nicotiana tabacum cultivar SR1 / Fatin Iffah Rasyiqah Mohamad Zoolkefli
title_sort effect of kdel and ppi genes co-expression on the anti-toxoplasma scfv activity in transgenic nicotiana tabacum cultivar sr1 / fatin iffah rasyiqah mohamad zoolkefli
publishDate 2018
url http://studentsrepo.um.edu.my/12033/1/Fatin_Iffah.pdf
http://studentsrepo.um.edu.my/12033/2/Fatin_Iffah_Rasyiqah.pdf
http://studentsrepo.um.edu.my/12033/
_version_ 1738506559389958144
score 13.211869

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