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. 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 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 1. Construction of recombinant plasmids ＆ HBcAg purification 2. In vitro transfections and Western blot analysis 3. Preparation of ABM 4. Immunization of mice and Measurement of antibody response 5. T cell proliferation assay

4 Materials and methods 1. Construction of recombinant plasmids ＆ HBcAg purification 2. In vitro transfections and Western blot analysis 3. Preparation of ABM 4. Immunization of mice and Measurement of antibody response 5. T cell proliferation assay

5 Method pBI1301-HBcAg （ BamHI 、 NotI ） pcDNA3 （ CMV promotor ） pET28a （ 6xHis tag ） BL21 （ DE3 ） pLysS 10mmol/l Tris-HCl Ni 2+ -NTA Sepharose 6B column

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

7 Materials and methods 1. Construction of recombinant plasmids ＆ HBcAg purification 2. In vitro transfections and Western blot analysis 3. Preparation of ABM 4. Immunization of mice and Measurement of antibody response 5. T cell proliferation assay

8 Expression of HBcAg in vitro pcDNA3 vectorpcDNA3-HBcAg

9 Materials and methods 1. Construction of recombinant plasmids ＆ HBcAg purification 2. In vitro transfections and Western blot analysis 3. Preparation of ABM 4. Immunization of mice and Measurement of antibody response 5. 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 1. Construction of recombinant plasmids ＆ HBcAg purification 2. In vitro transfections and Western blot analysis 3. Preparation of ABM 4. Immunization of mice and Measurement of antibody response 5. 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 Competitive ELISA HBcAg Anti-HBcAg antibody HRP-labeled anti-HBcAg antibody

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

15 Materials and methods 1. Construction of recombinant plasmids ＆ HBcAg purification 2. In vitro transfections and Western blot analysis 3. Preparation of ABM 4. Immunization of mice and Measurement of antibody response 5. 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

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 Discussion ＊ FMDV—foot-and-mouth disease virus

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] [2] [3] [4]Abul K. Abbas, Andrew H. Lichtman. Cellular and molecular immunology. 5th 13, 166. [5]Hugh B. Fackrell. ppt. Antigen-Antibody Interactions. [6] [7] [8] /pet.pdf [9] mages/infection.gif

25 The End