Vector Optimization for the High-Level Expression of Oplegnathus fasciatus Interferon-Gamma in Chlorella vulgaris

다국어 초록 (Multilingual Abstract)

Recombinant protein production in microalgae is emerging as a promising alternative to conventional host systems such as Escherichia coli, yeast, and mammalian cells, which are often limited by production scale, cost, or post- translational modification (PTM) limitations. Microalgae have been attracting attention as an oral vaccine delivery platform to address these challenges. Chlorella vulgaris is a strong candidate as an oral vaccine platform due to its Generally Recognized as Safe (GRAS) status, ease of cultivation at low cost, and amenable to post-translational modification. However, the low efficiency of recombinant protein expression through genetic transformation in C. vulgaris. In this study, we constructed and optimized an expression vector for the production of rock bream (Oplegnathus fasciatus) interferon gamma (IFN-γ) in C. vulgaris. We systematically compared various promoter- terminator combinations, including the CaMV 35S promoter and a salt inducible promoter (SIP) of C. vulgaris PKVL7422, the RBCS2 terminator and NOS terminator. Each vector was transformed into C. vulgaris PKVL7422. Protein expression levels in each C. vulgaris strain transformed with different vector constructs were assessed by SDS-PAGE and Western blotting. Our results showed that recombinant protein expression levels varied significantly depending on the promoter-terminator combination. In particular, the vector containing the SIP and NOS terminator, which were induced under salt stress conditions, showed the highest expression. The same results were obtained when analyzed by qRT-PCR of the interferon transcript. Optimization of SIP_IFN_NOS resulted in increased protein expression when treated with 250 mM NaCl and salt at the beginning of the culture. Interferon was expressed in a glycosylated state, as observed by band shifts observed with PNGase, O- glycosidase, and neuraminidase treatment. By optimizing the promoter-terminator combination in C. vulgaris PKVL7422, we were able to effectively enhance the expression of O. fasciatus IFN-γ, a key immune-related protein. Overall, this study presents a vector optimization strategy to enhance the efficiency of recombinant protein production in C. vulgaris, thereby contributing to the potential of a microalgae-based oral vaccine platform for aquaculture.

목차 (Table of Contents)

• Introduction 1
• Materials and Methods 5
• 1. Codon optimization and gene synthesis 5
• 2. Vector construction 6
• 3. PCR amplification of NR region for transformation 9
• 4. Culture conditions and transformation of C.vulgaris 12
• 5. Genomic DNA extraction and PCR confirmation 15
• 6. NaCl induction and culture conditions 17
• 7. SDS-PAGE and western blotting 18
• 8. Comparison of the effect of integration location on transformation
• efficiency and protein expression 19
• 9. Optimization of salt treatment concentration and duration 21
• 10. RNA extraction and cDNA synthesis 22
• 11. Quantitative real-time PCR (qRT-PCR) mRNA of 5 constructs 23
• 12. Production of polyclonal antibody for O. fasciants IFN-γ 25
• 13. Deglycosylation analysis of IFNγ_OF proteins 29
• Results 31
• 1. Transformant selection using ClO3 toxicity on C. vulgaris 31
• 2. Detection of IFNγ_OF genes in transformed colonies 33
• 3. Western blot analysis of IFNγ_OF protein from transformant by 5 constucts
• 35
• 4. Comparative evaluation of NR-Location variants on transformation
• efficiency 38
• 5. Optimum of expression conditions from the transformant by SIP/NOS 40
• 6. qRT-PCR analysis of IFNγ_OF gene from transformant by 5 constructs 45
• 7. Production IFNγ_OF polyclonal antibody for westren blot 46
• 8. Prediction and enzymatic verification of glycosylation in IFNγ_OF 49
• Discussion 52
• 국문 초록 58
• Acknowledgement 60
• Reference 61