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2003 中研院之行

2003 中研院之行. 趙裕展老師實驗室. 趙裕展老師. Expression of Spike Immunodominant Domains Using Baculovirus Expression Vector System. 蔡聖輝. Introduction. SARS-CoV. S - Spike protein: receptor binding, cell fusion, major antigen E - Envelope protein: small, envelope-associated protein

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2003 中研院之行

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  1. 2003中研院之行 趙裕展老師實驗室 趙裕展老師

  2. Expression of Spike Immunodominant Domains Using Baculovirus Expression Vector System 蔡聖輝

  3. Introduction

  4. SARS-CoV S - Spike protein: receptor binding, cell fusion, major antigen E - Envelope protein: small, envelope-associated protein M - Membrane protein: transmembrane - budding & envelope formation SARS: Severe Acute Respiratory Syndrom BEVS: Baculovirus Expression Vector System

  5. Introduction Spike protein is a membrane protein with a large extracellular domain, and is a major inducer of protective immune responses 200 a.a. Spike, SARS-CoV 450-657 equivalent to 417-547 of HCoV-229E coil-coiled oligomerization domain 547 763 966

  6. 1.Preparation of vABhRpS547H6 pABhRpS547H6 Linearize virus DNA co-transfection recombinant dead purify (End-point dilution)

  7. End-point dilution Wt：98 Wt：980 Recombinant virus：2 Recombinant virus：20 Wt：999 Dilution 10x Recombinant virus：1 Wt：100 1 Recombinant virus：0 50 1 1000 Wt：100 Recombinant virus：0

  8. End-point dilution Wt：7 Wt：700 Recombinant virus：3 Recombinant virus：300 Wt：980 Dilution 100x Recombinant virus：20 3 Wt：9 7 Recombinant virus：1 1 50 Wt：10 Recombinant virus：0

  9. End-point dilution Recombinant virus：1000 Recombinant virus：1 Wt：700 Dilution 1000x Recombinant virus：300 100% Wt：1 3 7 Wt：1

  10. Preparation of vABhRpS547H6 pABhRpS547H6 Linearize virus DNA co-transfection recombinant dead purify amplify test titer 4.21X108 pfu/ml

  11. Identification of expression of S547 1 2 3 - + PNGase A sample kDa Western blotting 90 S547(glycosylation) 70 55 antibodies against His-tag S547 + + - vABhRpS547H6 Signal peptide control medium medium marker His-tag S547= 60.784 kDa (theoretically) glycosylation

  12. 2.Construction of pABhRpS763rBH6 pABhRpS966rBH6 epithelial cells in upper respiratory tract Mucosal inductive sites ricinB Antigen-processing sIgA Receptor M RicinB binds the sugar moiety, which, in turn, leading to internalization by M-cells. M-cell is able to process antigen, resulting in the activation of mucosal immune response. And the hallmark of mucosal immunity is the formation of sIgA.

  13. Spike ricinB Since ricinB is able to target specific M-cells, any polypeptide (in our case, spike protein) fused to ricinB could be targeted and internalized into M-cells, resulting in development of mucosal immune response against SARS insults.

  14. procedure S763 S763 S966 Cla I Stu I S966 EcoR I PCR S763 EcoR I Cla I Stu I S966 Cla I EcoR I pBSrB ricinB H6 Cla I Stu I Pac I Sac I Sxxx EcoR I pBSrBSxxx ricinB H6 Pac I Sac I

  15. Stu I Sxxx pBSSxxxrB ricinB H6 Pac I Sac I digestion Ligation with pABhRpX p-polh Sxxx pABhRpS763rBH6 pABhRpS966rBH6

  16. Summary 1.Successful construction of recombinant virus vABhRpS547H6. 2. Another four viruses which contain S763 and S966 are currently under construction.

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