Coimmunization of Agaricus blazei Murill extract with hepatitis B virus core protein through DNA...
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Coimmunization of Agaricus bla zei Murill extract with hepati tis B virus core protein throu gh DNA vaccine enhances cellul ar and humoral immune response s L. Chen, H.J. Shao*, Y.B. Su The Key Laboratory of Cell Biology and Tumor Cell Engineerin g, School of Life Sciences, Xiamen University, Xiamen, PR Ch ina Received 3 November 2003; received in revised form 5 Decembe r 2003; accepted 29 December 2003 91390585-- 銘銘銘銘銘銘銘銘銘銘 -- 銘銘銘
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Transcript of Coimmunization of Agaricus blazei Murill extract with hepatitis B virus core protein through DNA...
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
