臺灣博碩士論文加值系統

登革熱病毒屬於黃質病毒科(Flaviviridae)黃質病毒屬(Flavivirus)。此屬病毒會造成許多人類的感染性疾病。在登革熱病毒產生蛋白質的過程，會先產生單一個蛋白質轉譯區，再切割成10個獨立的蛋白質。其中，NS2A、NS2B、NS4A及NS4B是較小的非結構性蛋白，具有疏水的特性，因此可能為和細胞膜有關的蛋白質。這四種蛋白質中，只有NS2B已知會參與蛋白切割酶反應，而NS2A、NS4A、NS4B尚未被鑑定出確切的生化功能。為研究登革熱病毒此四蛋白質的結構與功能，首先必須建構及表現此四種基因並對這四種蛋白質做功能性測試。本研究嘗試將這四種蛋白質在大腸桿菌和哺乳類細胞中，以加上HA-His (influenza hemagglutinin-polyhistidine protein)的融合蛋白(fusion protein)形式表現。為了在大腸桿菌表現系統中表現此四種蛋白質，遂將NS2A、2B、4A及4B的轉錄區域構築在修飾過的pET-30a(+)表現載體——pETΔ5T上。NS2B、4A及4B的HA-His融合蛋白可在大腸桿菌中，以西方墨點法測得。NS2A的轉錄產物則可以北方墨點法測得。NS2B、4A、4B在BHK-21以及NS2A在293T細胞中表現，也可以西方墨點法偵測。溶斑試驗(plaque assay)的結果顯示：表現NS2B的細胞經登革熱病毒感染後，會形成較對照組小的溶斑，而表現NS4A的細胞則會產生較大的溶斑。

Abstract (Chinese)……………………………………………………i
Abstract (English)……………………………………………………ii
Acknowledgement………………………………………………………iii
Contents…………………………………………………………………iv
Tables…………………………………………………………………viii
Figures…………………………………………………………………ix
Appendix…………………………………………………………………x

Chapter 1. Introduction……………………………………………………………1
1.1 Dengue Virus………………………………………………………1
1.2 Genome of Dengue virus…………………………………………2
1.3 Properties of viral proteins…………………………………2
1.4 Four small non-structural proteins of Dengue virus……3
1.5 Properties of NS2A………………………………………………4
1.6 Properties of NS2B………………………………………………5
1.7 Properties of NS4A………………………………………………6
1.8 Properties of NS4B………………………………………………7
1.9 The overview of experiment……………………………………8

Chapter 2. Materials and Methods…………………………………9
2.1 Materials……………………………………………………………9
2.1.1 Virus………………………………………………………………9
2.1.2 Cell lines………………………………………………………9
2.1.3 Bacterial strains………………………………………………9
2.1.4 Plasmids…………………………………………………………9
2.1.5 Primers…………………………………………………………10
2.1.6 Chemicals, enzymes, and reagents…………………………11
2.1.7 Antibodies………………………………………………………14
2.1.8 Kits………………………………………………………………15
2.1.9 Buffers…………………………………………………………15
2.1.10 Media……………………………………………………………17
2.1.11 Equipments……………………………………………………18
2.2 Methods……………………………………………………………19
2.2.1 Transformation of E. coli…………………………………19
2.2.1.1 Preparation of competent cells…………………………19
2.2.1.2 Transformation………………………………………………19
2.2.2 Plasmid DNA extraction………………………………………20
2.2.3 Restriction enzyme digestion………………………………20
2.2.4 Klenow enzyme fill in and blunt end ligation…………21
2.2.5 Cell culture……………………………………………………21
2.2.6 Tranfection of mammalian cell……………………………21
2.2.7 Expression of four nonstructural proteins in mammalian cell, BHK-21………………………………………………22
2.2.8 Plaque assay……………………………………………………22
2.2.9 Statistical analysis…………………………………………23
2.2.10 Expression of four nonstructural proteins in E. coli………………………………………………………………………23
2.2.11 Western blot analysis………………………………………23
2.2.12 RNA extraction………………………………………………24
2.2.13 Northern blot analysis……………………………………24

Chapter 3. Results……………………………………………………27
3.1 Construction of pcDNA3-D24B-HAHis expression plasmid…27
3.2 Sequence analysis of NS4B gene………………………………27
3.3 Construction of pcDNA3(pro)-D22A-HAHis, pcDNA3(pro)-D22B-HAHis, pcDNA3(pro)-D24A-HAHis, and pcDNA3(pro)-D24B-HAHis expression plasmids…………………………………………28
3.4 Construction of pETΔ5T-D22A-HAHis, pETΔ5T-D22B-HAHis, pETΔ5T- D24A-HAHis, and pETΔ5T-D24B-HAHis expression plasmids…………………………………………………………………29
3.4.1 Construction of pETΔ5T expression vector……………29
3.4.2 Construction of pETΔ5T-D22A-HAHis, pETΔ5T-D22B-HAHis, pETΔ 5T-D24A-HAHis, and pETΔ5T-D24B-HAHis expression plasmids…………………………………………………29
3.5 Expression of four nonstructural genes in mammalian cells……………………………………………………………………30
3.5.1 Expression of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pcDNA3-D24B-HAHis in BHK-21…………………………………30
3.5.2 Expression of pNS2A-HAHis in 293T………………………31
3.5.3 Expression of pcDNA3(pro)-D22A-HAHis, pcDNA3(pro)-D22B- HAHis, pcDNA3(pro)-D24A-HAHis, and pcDNA3(pro)-D24B-HAHis in BHK-21………………………………………………………32
3.6 Plaque assay of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pcDNA3-D24B-HAHis in BHK-21…………………………………32
3.7 Expression of four nonstructural genes in E. coli ……33
3.7.1 Expression of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pcDNA3-D24B-HAHis in E. coli…………………………………33
3.7.2 Expression of pcDNA3(pro)-D22A-HAHis, pcDNA3(pro)-D22B- HAHis, pcDNA3(pro)-D24A-HAHis, and pcDNA3(pro)-D24B-HAHis in E. coli………………………………………………………34
3.7.3 Expression of pETΔ5T-D22A-HAHis, pETΔ5T-D22B-HAHis, pETΔ5T-D24A-HAHis, and pETΔ5T-D24B-HAHis in E. coli………34
3.7.4 Detection of NS2A mRNA in E. coli………………………35


Chapter 4. Discussions………………………………………………36
4.1 Construction of pcDNA3-D24B-HAHis expression plasmid and sequence analysis………………………………………………36
4.2 Expression of four nonstructural proteins in mammalian cells……………………………………………………………………37
4.2.1 Expression of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pcDNA3-D24B-HAHis in BHK-21…………………………………37
4.2.2 Expression of pNS2A-HAHis in 293T………………………37
4.3 Plaque assay of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pcDNA3-D24B-HAHis in BHK-21…………………………………38
4.4 Expression of the four nonstructructural genes in E. coli………………………………………………………………………38
4.4.1 Expression of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pcDNA3-D24B-HAHis in E. coli…………………………………38
4.4.2 Construction of pcDNA3(pro)-D22A-HAHis, pcDNA3(pro)-D22B- HAHis, pcDNA3(pro)-D24A-HAHis, and pcDNA3(pro)-D24B-HAHis expression plasmids…………………………………………39
4.4.3 Expression of pcDNA3(pro)-D22A-HAHis, pcDNA3(pro)-D22B- HAHis, pcDNA3(pro)-D24A-HAHis, and pcDNA3(pro)-D24B-HAHis in E. coli and BHK-21………………………………………40
4.4.4 Construction of pETΔ5T-D22A-HAHis, pETΔ5T-D22B-HAHis, pETΔ 5T-D24A-HAHis, and pETΔ5T-D24B-HAHis expression plasmids…………………………………………………40
4.4.5 Expression of pETΔ5T-D22A-HAHis, pETΔ5T-D22B-HAHis, pETΔ 5T-D24A-HAHis, and pETΔ5T-D24B-HAHis in E. coli………………………………………………………………………41

Chapter 5. Conclusion………………………………………………42

Reference………………………………………………………………43


Tables
3.1 Contents of the PCR reaction…………………………………49
3.2 Condition for the PCR reaction of NS4B……………………49
3.3 Comparison of the NS4B sequence variations between Taiwan local strain PL046 and New Guinea C strain (Accession No. M29095)………………………………………………50
3.4 The number and size of plaques formed in the assay plates……………………………………………………………………51

Figures

1.1 World distribution of dengue viruses and their mosquito vector, Aedes aegypti, in 2005……………………………………52
1.2 Processing of Flavivirus polyprotein………………………52
1.3 Nucleotide mapping of NS2A, NS2B, NS4A and NS4B in dengue virus type 2 genome…………………………………………52
1.4 NS2B hydrophobicity plots and a hypothetical model of NS2B-NS3pro association with membranes…………………………53
1.5 The backbone structures of DEN2 NS3 protease with the cofactor, NS2B…………………………………………………………54
3.1 Restriction enzyme digestion of pcDNA3-4B-HAHis………55
3.2 Alignment of nucleotide sequences at NS4B of NGC and PL046 strain……………………………………………………………57
3.3 Maps of pcDNA3(pro)-D22A-HAHis, pcDNA3(pro)-D22B-HAHis, pcDNA3(pro)-D24A-HAHis, and pcDNA3(pro)-D24B-HAHis……………………………………………………………………58
3.4 Restriction enzyme digestion of pcDNA3(pro)-D22A-HAHis, pcDNA3(pro)-D22B-HAHis, and pcDNA3(pro)-D24B-HAHis…………59
3.5 Sequence analysis of pcDNA3(pro)-D24A-HAHis open reading frame…………………………………………………………61
3.6 Map of pETΔ5T……………………………………………………62
3.7 Restriction enzyme digestion of pETΔ5T…………………63
3.8 Sequence of blunt-end ligated region on pETΔ5T………63
3.9 Restriction enzyme digestion of pETΔ5T-D22A-HAHis, pETΔ5T-D22B- HAHis, pETΔ5T-D24A-HAHis, and pETΔ5T-D24B-HAHis.68
3.10 Coomassie blue staining and Western blot of nonstructural proteins expressed in BHK-21……………………69
3.11 Coomassie blue staining and Western blot of NS2A expressed in 293T……………………………………………………70
3.12 BHK-21 expressed nonstructural proteins analyzed by coomassie blue staining and Western blot………………………71
3.13 Plaque assay of BHK-21 transfected with different plasmids…………………………………………………………………72
3.14 Coomassie blue staining and Western blot of nonstructural proteins expressed in E. coli NovaBlue(DE3).73
3.15 E. coli NovaBlue(DE3) expressed nonstructural proteins analyzed by coomassie blue staining and Western blot………75
3.16 Coomassie blue staining and Western blot analyzing nonstructural proteins expressed in E. coli NovaBlue(DE3).77
3.17 Northern blot of NS2A mRNA expressed in E. coli NovaBlue(DE3)…………………………………………………………78
4.1 Restriction enzyme digestion of pNS4B-HAHis……………79

Appendix
Appendix 1 Map of pcDNA3(pro)-D34B-HAHis………………………80
Appendix 2 Map of pET-30a(+)………………………………………81
Appendix 3 Map of pBluescript II SK (+)………………………82
Appendix 4 Maps of pNS2A-HAHis, pNS2B-HAHis, pNS4A-HAHis, and pNS4B-HAHis………………………………………………………83

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