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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 responses.

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91390585-- 銘傳大學生物科技學系 -- 張倪綸

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  1. Coimmunization of Agaricus blazei Murill extract with hepatitis B virus core protein through DNA vaccine enhances cellular and humoral immune responses L. Chen, H.J. Shao*, Y.B. SuThe 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

  2. Introduction • ABM—Agaricus blazei Murill • HBcAg—Hepatitis B virus core antigen • DNA vaccine

  3. Materials and methods • Construction of recombinant plasmids ＆ HBcAg purification • In vitro transfections and Western blot analysis • Preparation of ABM • Immunization of mice and Measurement of antibody response • T cell proliferation assay

  4. Materials and methods • Construction of recombinant plasmids ＆ HBcAg purification • In vitro transfections and Western blot analysis • Preparation of ABM • Immunization of mice and Measurement of antibody response • T cell proliferation assay

  5. Method pBI1301-HBcAg （BamHI、NotI） pcDNA3 （CMV promotor） 10mmol/l Tris-HCl pET28a （6xHis tag） Ni2+-NTA Sepharose 6B column BL21（DE3）pLysS

  6. Expression of HBcAg in E.coil lane1：maker lane2：pET28a lane3：pET28a-HBcAg

  7. Materials and methods • Construction of recombinant plasmids ＆ HBcAg purification • In vitro transfections and Western blot analysis • Preparation of ABM • Immunization of mice and Measurement of antibody response • T cell proliferation assay

  8. Expression of HBcAg in vitro pcDNA3 vector pcDNA3-HBcAg

  9. Materials and methods • Construction of recombinant plasmids ＆ HBcAg purification • In vitro transfections and Western blot analysis • Preparation of ABM • Immunization of mice and Measurement of antibody response • T cell proliferation assay

  10. ABM extract Extracted with boiled water freeze-dried dissolved in 0.9%NaCl（10mg/ml） Filtered through a 0.45um membrane filter

  11. Materials and methods • Construction of recombinant plasmids ＆ HBcAg purification • In vitro transfections and Western blot analysis • Preparation of ABM • Immunization of mice and Measurement of antibody response • T cell proliferation assay

  12. 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

  13. Anti-HBcAg antibody HRP-labeled anti-HBcAg antibody HBcAg Competitive ELISA

  14. Antibody response pcDNA3 ABM extract pcDNA3-HBcAg pcDNA3-HBcAg plus ABM extract

  15. Materials and methods • Construction of recombinant plasmids ＆ HBcAg purification • In vitro transfections and Western blot analysis • Preparation of ABM • Immunization of mice and Measurement of antibody response • T cell proliferation assay

  16. Method Digested with trypsin for 2 h Remove red blood cells T cells were grown in 96-well plates incubated with purified HBcAg

  17. T cell proliferation response Concanavalin A 100μg 100μg 100μg 100μg

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

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

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

  21. ＊FMDV—foot-and-mouth disease virus Discussion

  22. Discussion

  23. 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.

  24. 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

  25. The End

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