Development of transformation system of Dunaliella salina for the expression of viral hemorrhagic septicemia virus glycoprotein and flounder growth hormone
- Abstract
- Eschrichia coli has been used as the primary system for the production of recombinant protein. However, it has some limitation such as formation of insoluble inclusion body, toxicity of expressed foreign protein and lack of post-translational modification. Eukaryotic expression system such as yeast or cultured cell can be used but the cost for culture and purification is relatively high. In contrast, microalgae such as Dunaliella salina that used in this study can be another alternative for eukaryotic expression system. We have developed an efficient system for the transformation of D. salina and tested expression of two foreign proteins. In this system we knocked out the wild type nitrate reductase (NR) gene of D. salina that can convert chlorate to toxic chlorite in addition to its normal reduction of nitrate to nitrite. The target DNA containing the cauliflower mosaic virus 35S promoter- foreign gene-transcription terminator of rbcs2 was flanked by DNA fragment form D. salina NR gene. The target DNA introduced into D. salina cell by glass bead vortexing methods replaced the wild type NR gene by homologous recombination and resulted in transformants that can grow on selection medium containing 100 mM KClO3. We used the glycoprotein gene of viral hemorrhagic septicemia virus (VHSV) and flounder growth hormone gene as target as target genes. Selected transformants were analyzed by PCR to investigate integration of introduced DNA into D. salina genome. Western blot analysis with polyclonal antibody against target protein confirmed the expression of these target proteins in transformed D. salina, which can be used as bioreactor for recombinant protein after futher refinement.
- Issued Date
- 2017
- Awarded Date
- 2017. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 미생물학과
- Advisor
- 최태진
- Table Of Contents
- Contents
Abstract ⅴ
Introduction 1
Materials and Methods 4
fGH codon optimization 4
Escherichia coli transformation and confirmation of the G gene cDNA sequence 4
Construction of plasmids and preparation of inclusion bodies 5
Construction of vector for transforming D. salina 6
Determining of protein concentration 9
Preparation of dialysis tubing 9
First antibody production 9
Microalgae strain and culture condition 11
Preparation of high-concentration plasmid DNA 11
Transformation using glass beads 12
Screening of transformants 12
DNA extraction and PCR 12
Western blot analysis 15
Results 16
Confirmation of overexpression in transformed E. coli cells 16
Confirmation of VHSV G protein expression in E. coli cells 18
Construction of plasmid vector, pSK-HR-vG and fGH_opti, for transformation in
D. salina 20
Screening of the transformed D. salina on selective media 23
Detection of the gene of interest that is successfully integrated in D. salina NR gene by homologous recombination using PCR 25
Western blot analysis for detecting VHSV G protein and fGH protein expression in D. salina 27
Discussion 29
국문초록 32
References 34
List of Figures
Figure 1. Conformation of overexpression in transformed E. coli cells was performed using antibody and assessed using SDS-PAGE 17
Figure 2. Detection of G polyclonal antibody on the PVDF membrane using western blot analysis 19
Figure 3. Diagram of plasmid constructs 21
Figure 4. Detection of G and optimized fGH DNAs were digested with BamHI and XhoI 22
Figure 5. Comparison of the differences in D. salina growth on media 24
Figure 6. Detection of integrated DNA target site using specific PCR primer sets for
different regions 26
Figure 7. Western blot analysis of VHSV G protein and fGH protein expressed in transformed D. salina 28
List of Tables
Table 1. Primers used for the PCR amplification of G gene 8
Table 2. Primers used in the identified of NR gene expression cassette 14
- Degree
- Master
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