臺灣博碩士論文加值系統

登革熱病毒是屬於黃熱病毒屬裡的病毒，會造成許多人類的傳染性疾病。在病毒蛋白質的產生過程，會先產生單一個蛋白質轉譯區域，再切割成11個各別的蛋白質，其中，NS2A、NS2B、NS4A及NS4B這四個蛋白質是比較小的非結構性蛋白，擁有疏水性的特性，並且可能為和膜有關的蛋白質。這四個蛋白質中，只有NS2B的功能已知，被認為是和病毒的蛋白質切割酵素NS3有關聯，其他NS2A、NS4A及NS4B的特定生化功能都還沒有被發現。因此，在有興趣研究這四個蛋白的結構及功能的情況下，首先要做的就是轉殖並表現這四個基因。這四個基因在大腸桿菌，真菌(P. pastoris)，及哺乳類細胞中，以加上His-HA(polyhistidine-influenza hemagglutinin protein)的融合蛋白(fusion protein)形式表現。為了在不同的表現系統表現這些蛋白質，NS2A、NS2B、NS4A及NS4B的轉錄區域藉著RT-PCR由登革熱病毒(PL046 strain)大量複製後，成功的轉殖到大腸桿菌及哺乳類細胞的共同表現載體：pcDNA3，及P. pastoris表現載體：pPIC3.5。加上HA-His的NS2A、NS2B、NS4A及NS4B重組蛋白，可以在大腸桿菌的上清液(soluble fraction)以西方墨點法測得，並可以分離管柱(nickel column)加以純化。在哺乳類細胞表現的NS2A、NS2B、及NS4A重組蛋白及在P. pastoris (KM71 strain)表現的NS2B及NS4A均可以西方墨點法測得。

Dengue virus is a member of flaviviridas, which cause many infectious diseases of human. Dengue virus encodes 11 proteins in a single open reading frame. Among them, NS2A, 2B, 4A and 4B are small non-structural proteins exhibiting conserved hydrophobicity profiles among flaviviuses, suggesting their roles as membrane-associated proteins. Only NS2B has been suggested to be involved in protease activity. Specific biological functions for NS2A, 4A and 4B have not been identified. To delineate the structure and function of these four proteins, I ttempted to establish cloning and expression of these four genes. They were initially expressed as His-HA (polyhistidine-influenza hemagglutinin protein) fusion proteins in E. coli, Pichia pastoris, and mammalian cells. For expression of these proteins in different expression systems, the coding region of NS2A, 2B, 4A and 4B were RT-PCR amplified from dengue virus type 2 PL-046 strain and were successfully cloned into pcDNA3, an E. coli/mammalian cells expression shuttle vector, and pPIC3.5, a Pichia pastoris expression vector. Recombinant proteins of NS2A, 2B, 4A and 4B fusing with HA-His tag can be detected in soluble fraction of E. coli expression system by Western blotting analysis and purified with nickel column. Protein expression of NS2A, 2B and 4A in mammalian cell and NS2B and NS4A in Pichia pastoris, KM71 was also detected by Western analysis.

Contents
Abstract (Chinese) …………………………………………… i
Abstract (English) …………………………………………… ii
Acknowledgement …………………………………………… iii
Contents …………………………………………………… iv
Contents (Table) …………………………………………… vi
Contents (Figure) …………………………………………… vii
Chapter 1. Introduction
1.1 Dengue virus ………………………………………… 1
1.2 Genome of dengue virus…………………………… 2
1.3 Properties of viral protein……………………… 2
1.4 Four small non-structural proteins of dengue virus…… 3
1.5 Properties of NS2A………………………………… 3
1.6 Properties of NS2B………………………………… 5
1.7 Properties of NS4A………………………………… 6
1.8 Properties of NS4B………………………………… 7
1.9 The Pichia pastoris expression system………… 7
1.10 The purpose of experiment………………………… 8
Chapter 2. Materials and Methods
2.1 Materials………………………………………… 10
2.2 Isolation of Virus RNA…………………………… 11
2.3 Reverse Transcription-Polymerase Chain Reaction……… 11
2.4 Construction of expression vectors for the E. coli and mammalian cell………………………………………… 12
2.5 Construction of expression vectors for Pichia pastoris expression system…………………………………… 13
2.6 Expression of four recombinant nonstructural proteins in E. coli…………………………………………………… 14
2.7 SDS-PAGE and Western blotting analysis………… 15
2.8 Purification of recombinant nonstructural proteins from E. coli……………………………………………… 16
2.9 Transfection of BHK-21 cells by Lipofetamine reagent…………………………………………………… 16
2.10 Transformation of Pichi pastoris………………… 17
2.11 Screening transformants for positive clones……18
2.12 Expression of four nonstructural proteins in Pichi pastoris………………………………………………… 18
Chapter 3. Results
3.1 Construction of the four nonstructural genes…………20
3.2 Sequencing analysis of four nonstructural proteins…20
3.3 Expression of four nonstructural proteins in E. coli…… 21
3.4 Expression of four nonstructural proteins in mammalian cells, BHK-21…………………………………………22
3.5 Purification of nonstructural proteins NS2A-His, NS2B-His, NS4A-His and NS4B-His from E.coli……… 23
3.6 Screening transformants for positive clones by PCR…… 24
3.7 Expression of four nonstructural proteins in P. pastoris… 25
Chapter 4. Discussion
4.1 Cloning and sequence analyzing……………………27
4.2 Expression and purification of four nonstructural proteins in E. coli……………………………………… 27
4.3 Expression of four nonstructural proteins in mammalian cells, BHK-21……………………………………………30
4.4 Expression of four nonstructural proteins in yeast, P. pastoris………………………………………………… 30
Chapter 4. Conclusion………………………………………………33
Reference ………………………………………………………… 56
Appendix ………………………………………………………… 62

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