利用哺乳動物細胞表現嚴重急性呼吸道症候群冠狀病毒之棘蛋白片段

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研究生：	張珈尹 Chia-Yin Chang
論文名稱：	利用哺乳動物細胞表現嚴重急性呼吸道症候群冠狀病毒之棘蛋白片段 Production of Truncated Severe Acute Respiratory Syndrome Coronavirus Spike Protein by Mammalian Cell Expression System
指導教授：	吳夙欽 Suh-Chin Wu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	96
中文關鍵詞：	嚴重急性呼吸道症候群、冠狀病毒、疫苗、哺乳動物細胞、醣蛋白、基因放大、棘蛋白
外文關鍵詞：	severe acute respiratory syndrome, coronavirus, SARS, CHO, Endo H, exon splicing enhancers, intron, DHFR, dihydrofolate reductase, methotrexate, MTX, ESE
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嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)的套膜Spike蛋白(S蛋白)在疫苗的研發上扮演了很重要的角色。截短的SARS-CoV TW1 病毒株之S蛋白 ─「SNHRI」(包含有三個S蛋白片段：S74-253、S294-739和S1129-1255，分子量為88 kDa)可在哺乳動物細胞中被表現出來。在哺乳動物細胞表現載體中加入一個138bp的intron可以分別在CHO/dhFr-細胞、Vero E6細胞和QBI-293A細胞中提升SNHRI蛋白的表現量達4倍、1.9倍和2.5倍。近一步的研究發現，由哺乳動物細胞表現出來的SNHRI蛋白主要是以對Endo H酵素敏感的醣蛋白形式(~115 kDa)存在，但CHO/dhFr-細胞及Vero E6細胞亦可製造少量可資抵抗Endo H酵素的 ~130 kDa SNHRI蛋白。在哺乳動物細胞表現載體中加入兩種Exon splicing enhancer (ESE)： Bidirectional splicing enhancer (BSE) 以及fibronectin EDA exon中的ESE (EDA ESE)可在CHO/dhFr-細胞中比無intron載體之SNHRI表現量提高1.7倍或2.6倍。當同時加入ESE和intron時，intron之提升SNHRI蛋白表現量的能力反而受到些許的抑制。為了建立可穩定大量表現SNHRI蛋白之CHO細胞，含有接在internal ribosome entry site後方之dihydrofolate reductase (dhfr)基因的兩個SNHRI表現載體(pSID與pISID)被建構出來，其中的pISID載體中含有一個138bp的intron。為了增加SNHRI蛋白在細胞株中的表現量，逐步提高之Methotrexate (MTX)濃度被用來篩選以pSID與pISID transfect的CHO/dhFr-細胞並引導SNHRI之基因在染色體中增加。兩種不同分子量之 SNHRI醣蛋白(~98與~115 kDa)可被穩定表現SNHRI的單一細胞株製造出來。其中，十二個穩定表現~115 kDa SNHRI蛋白的細胞株被挑選出來作進一步的基因放大。這份研究可作為日後發展哺乳動物細胞SARS-CoV單元疫苗之重要參考。

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. The truncated SARS-CoV TW1 S protein, SNHRI (88 kDa), carrying three S fragments (S74-253, S294-739, and S1129-1255) was expressed in mammalian cells. Mammalian cell expression of SNHRI glycoprotein with the use of a 138bp intron was found to increase by 1.9 folds, 2.5 folds, and 4.1 folds in Vero E6, QBI-293A cells, and CHO/dhFr- cells (dihydrofolate reductase [dhfr] gene deficient CHO cells), respectively. The expressed SNHRI glycoprotein was mainly Endo H-sensitive (~115 kDa glycoproteins) in these three mammalian cell lines. A minor form of Endo H-resistant glycoproteins (~130 kDa) was also found in CHO/dhFr- cells and VeroE6 cells. Expression vectors using the exon splicing enhancers, such as bidirectional splicing enhancer (BSE) or an exon splicing enhancer derived from the EDA alternative exon of the fibronectin gene (EDA ESE), was also found to increase SNHRI protein expression by 1.7 and 2.6 folds as compared to the intronless expression vector. Coupling BSE or EDA ESE with the 138bp intron leaded to suppression of the intron-enhancing effect. In order to establish stably expressing CHO cell clones, a stepwise methotrexate (MTX) selection was conducted in the CHO/dhFr- cells transfected with two amplifiable expression vectors, pSID (without intron) and pISID (with intron), containing a dhfr gene placed after an internal ribosome entry sites. Twelve stable cell clones expressing higher amount of ~115 kDa SNHRI protein were selected for gene amplification. This study provides useful information for future development of mammalian cell based SARS-CoV subunit vaccines.

Chinese abstract
English abstract
Acknowledgments
Content
   Introduction
1.    Severe Acute Respiratory Syndrome Coronavirus
1.1.    Epidemiology of SARS-CoV
1.2.    Basic Virology of SARS-CoV
1.3.    The Genome and Virus Structure of SARS-CoV
1.4.    SARS-CoV Spike Protein
1.5.    Binding of SARS-CoV Spike Protein to Receptors
1.6.    Spike Protein as Target for SARS-CoV Vaccine Development
2.    Mammalian Cell Expression System
2.1.    Overview of Mammalian Cell Expression Systems
2.2.    CHO/dhFr- Cell Expression Systems
2.3.    Dihydrofolate Reductase Gene Amplification
3.    The Influence of Intron and Exon Splicing Enhancers on Eukaryotic Gene Expression
3.1.    The Common Structure of an Eukaryotic Intron
3.2.    Overview of The Influence of Intron on Protein Expression
3.2.1    Splicing and Exon Junction Complex Formation
3.2.2    The Influence of Intron on mRNA Transcription
3.2.3    The Influence of Intron on Poly(A)-tail Addition
3.2.4    Effects of Splicing on mRNA Export
3.2.5    The Relationship Between Intron and Translation
3.2.6    The Difference of Intron Dependence and Enhancing effect among various gene and cell lines
3.3.    Overview of the Influence of Exon Splicing Enhancer on Protein Expression
3.3.1    The Exon Splicing Enhancer of Fibronectin EDA Exon
3.3.2    Bidirectional Splicing Enhancer
   Materials and Methods
1.    Cell Lines and Mediums
2.    Vectors Construction
2.1.    S NHRI
2.2.    Intron, Bidirectional splicing enhancer, and Exon splicing enhancer
2.3.    Expression Vector Construction for DHFR/MTX Gene Amplification
3.    Transient Expression in Mammalian Cells
4.    Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
5.    PNGase F Treatment of SNHRI Protein
6.    Endo H Treatment of SNHRI Protein
7.    Selection and Gene Amplification of Clones that Can Stably Express SNHRI Protein
   Results
1.    SNHRI and SFL Construction and Expression
2.    Optimization of SNHRI Expression with Intron and Exon Splicing Enhancers
2.1.    SNHRI Expression Efficiency in Different Cell Lines During the Presence or Absence of an Intron
2.2.    Glycosylation of SNHRI Expressed in CHO/dhFr- Cells by Intron Containing Construct
2.3.    The Enhancing Effect of EDA ESE and BSE in SNHRI Expression During the Presence or Absence of an Intron
3.    DHFR/MTX Gene Amplification for Establishing Stable CHO Cell clones that Express SNHRI Protein
   Discussion
1.    SARS-CoV SNHRI & SFL Protein Expression in CHO/dhFr- Cells
2.    Intron Addition for SNHRI Mammalian Cell Expression
3.    Exon Splicing Enhancers for SNHRI Expression in CHO/dhFr- Cells
4.    Glycosylation Patterns of SNHRI Produced in CHO/dhFr- Cells
5.    Conclusion
   Appendix
1.    The DNA Sequence of SFL
2.    The Protein Sequence of SFL
3.    The DNA Sequence of SNHRI
4.    The Protein Sequence of SNHRI
Appendix Table 1. The putative N-linked glycosylation sites of SFL and SNHRI
Appendix Figure 1. Selection of SNHRI expressing SID cell clones
Appendix Figure 2. Selection of SNHRI expressing ISID cell clones
Figures
Fig.1.    The genome and virus structure of SARS-CoV
Fig.2.    SARS-CoV Spike protein
Fig.3.    Dihydrofolate reductase (DHFR) related metabolism pathway
Fig.4.    The flowchart of establishing amplified stable cell clones expressing recombinant proteins in CHO/dhfr- expression system
Fig.5.    Construction and CHO/dhFr- cell transient expression of pSFL and pS vectors
Fig.6.    The influence of intron addition on SNHRI expression
Fig.7.    The N-linked glycosylation pattern of SNHRI protein
Fig.8.    Analysis of SNHRI expression efficiency when BSE or EDA ESE was placed in SNHRI open reading frame
Fig.9.    Analysis of SNHRI expression efficiency when EDA ESE was placed in the 5’-UTR of SNHRI expressing vectors
Fig.10.    Construction of amplifiable vectors, pSID and pISID
Fig.11.    SNHRI expression in selected SID and ISID cell clones
Fig.12.    Schematic figure of SID stable clone selection and gene amplification
Fig.13.    Schematic figure of ISID stable clone selection and gene amplification
Fig.14.    MTX selection process could lead to higher SNHRI expression
Fig.15.    The SNHRI expression efficiency of selected cell clones after 0.02uM MTX selections
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