20032003 中研院之行中研院之行趙裕展老師實驗室趙裕展老師實驗室

趙裕展老師趙裕展老師

Expression of Spike Immunodominant Domains Expression of Spike Immunodominant Domains Using Baculovirus Expression Vector SystemUsing Baculovirus Expression Vector System

蔡聖輝蔡聖輝

IntroductionIntroduction

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 SyndromBEVS: Baculovirus Expression Vector System

SARS-CoVSARS-CoV

IntroductionIntroduction

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-coiledoligomerization domain547

763966

1.Preparation of vABhRpS1.Preparation of vABhRpS547547HH66

pABhRpS547H6

Linearize virus DNA

co-transfection

recombinantdead

purify

(End-point dilution)

End-point dilutionEnd-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

End-point dilutionEnd-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

End-point dilutionEnd-point dilution

Wt ： 700

Recombinant virus ： 300

Dilution 1000x

Recombinant virus ： 1

Wt ： 1

Recombinant virus ： 1000

Wt ： 1

100%

7

3

Preparation of vABhRpSPreparation of vABhRpS547547HH66

pABhRpS547H6

Linearize virus DNA

co-transfection

recombinantdead

purify

amplify

test titer

4.21X108 pfu/ml

Identification of expression of SIdentification of expression of S547547

mar

ker

cont

rol

med

ium

S547(glycosylation) 70

med

ium

+ + -vABhRpSvABhRpS547547HH66

kDa

55

90

1 2 3

- +PNGase A

sample

Western blotting

antibodies against His-tag

S547

glycosylation

S547= 60.784 kDa(theoretically)

His-tagSignal peptide

epithelial cells in upper respiratory tractMucosal inductive sites

M

Antigen-processing

sIgA

Receptor

2.Construction of pABhRpS2.Construction of pABhRpS763763rBHrBH66

pABhRpS pABhRpS966966rBHrBH66

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.

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.

S763

S966

PCR

S763

S966

Cla I Stu I EcoR I

S763

S966

Cla I Stu I EcoR I

pBSrB

ricinB

Pac I Sac I

Cla I EcoR I

H6

pBSrBSxxx

ricinB

Pac I Sac I

EcoR I

H6

Sxxx

Cla I Stu I

procedureprocedure

pBSSxxxrB

ricinB

Pac I Sac I

H6

Sxxx

Stu I

digestionLigation with pABhRpX

p-polh

pABhRpS763rBH6

pABhRpS966rBH6

Sxxx

Summary

1.Successful construction of recombinant virus vABhRpSvABhRpS547547HH66..

2. Another four viruses which contain 2. Another four viruses which contain S763 and S966 are currently under construction.