91390585-- 銘傳大學生物科技學系 -- 張倪綸

Coimmunization of Agaricus blazei Murill extract with hepatitis B virus core protein through DNA vaccine enhances cellular and humoral immune respons

esL. Chen, H.J. Shao*, Y.B. Su

The Key Laboratory of Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, PR China

Received 3 November 2003; received in revised form 5 December 2003; accepted 29 December 2003

91390585-- 銘傳大學生物科技學系 --張倪綸

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Introduction

ABM—Agaricus blazei Murill HBcAg—Hepatitis B virus core antigen DNA vaccine

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Materials and methods

1. Construction of recombinant plasmids ＆ HBcAg purification

2. In vitro transfections and Western blot analysis

3. Preparation of ABM4. Immunization of mice and Measureme

nt of antibody response5. T cell proliferation assay

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Materials and methods

1. Construction of recombinant plasmids ＆ HBcAg purification

2. In vitro transfections and Western blot analysis

3. Preparation of ABM4. Immunization of mice and Measureme

nt of antibody response5. T cell proliferation assay

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Method

pBI1301-HBcAg（ BamHI 、 NotI）

pcDNA3（ CMV promotor）

pET28a（ 6xHis tag）

BL21 （ DE3 ） pLysS

10mmol/l Tris-HCl

Ni2+-NTASepharose 6B column

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Expression of HBcAg in E.coil

lane1 ： maker

lane2 ： pET28a

lane3 ： pET28a-HBcAg

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Materials and methods

1. Construction of recombinant plasmids ＆ HBcAg purification

2. In vitro transfections and Western blot analysis

3. Preparation of ABM4. Immunization of mice and Measureme

nt of antibody response5. T cell proliferation assay

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Expression of HBcAg in vitro

pcDNA3 vector pcDNA3-HBcAg

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Materials and methods

1. Construction of recombinant plasmids ＆ HBcAg purification

2. In vitro transfections and Western blot analysis

3. Preparation of ABM4. Immunization of mice and Measureme

nt of antibody response5. T cell proliferation assay

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ABM extract

Extracted with boiled water

freeze-dried

dissolved in 0.9%NaCl （ 10mg/ml ）

Filtered through a 0.45um membrane filter

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Materials and methods

1. Construction of recombinant plasmids ＆ HBcAg purification

2. In vitro transfections and Western blot analysis

3. Preparation of ABM4. Immunization of mice and Measureme

nt of antibody response5. T cell proliferation assay

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Method

Male BALA/c mice

Inject in left hind thigh muscles with 100ug of plasmid DNA in 0.9% NaCl

Boost 2 weeks

Competitive ELISA

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Competitive ELISA

HBcAg

Anti-HBcAg antibody

HRP-labeled anti-HBcAg antibody

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Antibody response

pcDNA3

pcDNA3-HBcAg

pcDNA3-HBcAg plus ABM extract

ABM extract

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Materials and methods

1. Construction of recombinant plasmids ＆ HBcAg purification

2. In vitro transfections and Western blot analysis

3. Preparation of ABM4. Immunization of mice and Measureme

nt of antibody response5. T cell proliferation assay

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Method

Digested with trypsin for 2 h

Remove red blood cells

T cells were grown in 96-well plates

incubated with purified HBcAg

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T cell proliferation response

Concanavalin A

100μg

100μg100μg

100μg

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Discussion

Of all fungi, ABM is particularly rich in polysaccharides associated with antitumoral activity.

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Discussion

After the secondary injection, coimmunization with ABM extract caused three- to fourfold increase of HBcAg antibody compared to HBcAg alone.

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Discussion

ABM extract can modulate the immune responses for foot-and-mouth disease virus (FMDV) DNA vaccine.

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Discussion

＊ FMDV—foot-and-mouth disease virus

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Discussion

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Conclusion

In general, the antibody response induced by the DNA vaccine is lower than that of the corresponding protein-based vaccine. Thus, improving the efficacy of DNA vaccine has become a critical issue.

ABM extract possesses adjuvant activity for the DNA vaccine.

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References

[1]http://agbio.coa.gov.tw/research_detail.asp?dno=8617&ito=35 [2]http://rcocc.csmu.edu.tw/database/b-23.PDF [3]http://tw.knowledge.yahoo.com/question/?qid=1305082907698 [4]Abul K. Abbas, Andrew H. Lichtman. Cellular and molecular immunology. 5th 13, 166. [5]Hugh B. Fackrell. ppt. Antigen-Antibody Interactions. [6]http://biotech.nsc.gov.tw/6-01-3.html [7] http://textbookofbacteriology.net/Secondaryresponse.jpeg [8]http://www.fhcrc.org/science/labs/hahn/methods/biochem_meth /pet.pdf [9]http://www.umanitoba.ca/faculties/medicine/units/immunology/I mages/infection.gif

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The End