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.
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