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. 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 SARS-CoV

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. 450-657 equivalent to 417-547 of HCoV-229E Spike, SARS-CoV coil-coiled oligomerization domain 547 763 966

6. 1.Preparation of vABhRpS 547 H 6 pABhRpS 547 H 6 Linearize virus DNA co-transfection recombinant dead purify (End-point dilution)

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

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

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

10. Preparation of vABhRpS 547 H 6 pABhRpS 547 H 6 Linearize virus DNA co-transfection recombinant dead purify amplify test titer 4.21X10 8 pfu/ml

11. Identification of expression of S 547 marker control medium S 547 (glycosylation) 70 medium + + - vABhRpS 547 H 6 kDa 55 90 1 2 3 - + PNGase A sample Western blotting antibodies against His-tag S 547 glycosylation S 547 = 60.784 kDa (theoretically) His-tag Signal peptide

12. epithelial cells in upper respiratory tract Mucosal inductive sites M Antigen-processing sIgA R eceptor 2.Construction of pABhRpS 763 rBH 6 pABhRpS 966 rBH 6 ricinB 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. ricinB Spike 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. S 763 S 966 PCR S 763 S 966 Cla I Stu I EcoR I S 763 S 966 Cla I Stu I EcoR I pBSrB ricinB Pac I Sac I Cla I EcoR I H6H6 pBSrBS xxx ricinB Pac I Sac I EcoR I H6H6 S xxx Cla I Stu I procedure

15. pBSS xxx rB ricinB Pac I Sac I H6H6 S xxx Stu I digestion Ligation with pABhRpX p-polh pABhRpS 763 rBH 6 pABhRpS 966 rBH 6 S xxx

16. Summary vABhRpS 547 H 6. 1.Successful construction of recombinant virus vABhRpS 547 H 6. 2. Another four viruses which contain 2. Another four viruses which contain S 763 and S 966 are currently under construction.