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目的研究茯苓多糖(PCP)-Ⅰ对乙肝疫苗抗原免疫小鼠免疫反应的影响。方法采用60℃水提、醇沉、透析和冷冻干燥获得茯苓粗多糖PCP。PCP再经过DEAE-纤维素和Sephadex G-100柱色谱分离,获得相对分子质量均一多糖PCP-Ⅰ。凝胶过滤色谱法测定其峰高相对分子质量,毛细管电泳法测定其单糖组成。乙肝疫苗抗原(HBs Ag,2μg/小鼠)分别与2个剂量PCP-Ⅰ(200μg/小鼠和50μg/小鼠)联用,肌内注射途径免疫小鼠2次,分别于初次免疫后14 d和第2次免疫后14、30、45、60和90 d,采用ELISA法测定小鼠血清特异性抗体Ig G、Ig M、Ig A及Ig G亚类抗体滴度水平。结果 PCP-Ⅰ为相对分子质量均一多糖,峰高相对分子质量约为3.0×104,单糖摩尔比为岩藻糖∶甘露糖∶葡萄糖∶半乳糖=1.00∶1.81∶0.27∶7.27。佐剂活性结果表明,在第2次免疫后14~90 d期间,与单独抗原组相比,联用50或200μg PCP-I均能显著提高受免小鼠血清特异性抗体滴度水平。单独抗原免疫产生的抗体类型主要为Ig G1和Ig M,而联用PCP-I则还产生高滴度的Ig G2a、Ig G2b和Ig G3。PCP-I对乙肝抗原佐剂效果明显优于铝佐剂,而且随着时间的延长,抗体水平变化不明显。PCP-I还能促进免疫小鼠脾T细胞和B细胞增殖,升高CD3+/CD19+的比值,降低CD4+/CD8+的比值。结论茯苓多糖PCP-I能显著提高乙肝抗原免疫小鼠体液免疫和细胞免疫能力,效果优于铝佐剂。
Objective To study the effect of tuckahoe polysaccharide (PCP) -Ⅰ on immune response in mice immunized with hepatitis B vaccine antigen. Methods Porcine polysaccharide PCP was obtained by water-extraction, alcohol precipitation, dialysis and freeze-drying at 60 ℃. PCP and then by DEAE-cellulose and Sephadex G-100 column chromatography to obtain a relatively homogeneous molecular weight polysaccharide PCP-Ⅰ. Gel filtration chromatography was used to determine the peak relative molecular mass. The monosaccharide composition was determined by capillary electrophoresis. Hepatitis B vaccine antigen (HBsAg, 2μg / mouse) were used in combination with 2 doses of PCP-Ⅰ (200μg / mouse and 50μg / mouse) respectively. The mice were immunized twice by intramuscular injection. d, and 14, 30, 45, 60 and 90 days after the second immunization. The titer of Ig G, Ig M, Ig A and Ig G subgroup antibodies of the serum-specific antibodies was measured by ELISA. Results PCP-Ⅰ was a homogeneous polysaccharide with molecular weight of about 3.0 × 104. The molar ratio of monosaccharides was fucose: mannose: glucose: galactose = 1.00:1.81:0.27:7.27. The results of the adjuvant activity showed that in the 14 to 90 days after the second immunization, 50 or 200 μg PCP-I in combination with the antigen alone group significantly increased the serum-specific antibody titer of the immunized mice. Antibodies produced by antigen alone immunized mainly IgG1 and IgM, while PCP-I in combination produced high titers of IgG2a, IgG2b and IgG3. The effect of PCP-I on hepatitis B antigen adjuvant was significantly better than that of aluminum adjuvant, and the antibody level did not change significantly with time. PCP-I can also promote the proliferation of splenic T lymphocytes and B cells in mice, increase the ratio of CD3 + / CD19 +, and decrease the ratio of CD4 + / CD8 +. Conclusion Pachymanin PCP-I can significantly improve humoral and cellular immune responses of mice immunized with hepatitis B antigen, which is superior to aluminum adjuvant.