PTD-HBcAg融合蛋白诱导特异性CTL抑制转基因小鼠HBV复制

Protein transduction domainhepatitis B virus core antigen fusion protein-induced specific cytotoxic T lymphocyte response inhibits hepatitis B virus replication in hepatitis B virus-transgenic mice

目的:探讨经PTD-HBcAg融合蛋白体内诱导的特异性细胞毒T淋巴细胞(CTL)对HBV转基因小鼠病毒的抑制作用.方法:20只HBV转基因小鼠随机分组,融合蛋白PTD-HBcAg及对照蛋白HBcAg经皮下免疫小鼠,每周1次,共3次.流式细胞仪检测脾细胞中胞内细胞因子水平;微粒子酶免疫分析法(MEIA)检测血清中乙型肝炎表面抗原(HBsAg)水平;荧光定量聚合酶链反应(PCR)检测HBVDNA水平;肝脏HE染色及免疫组织化学方法检测HBsAg表达.结果:PTD-HBcAg融合蛋白免疫转基因小鼠后,能有效上调特异性CTL数量,肝组织中炎性细胞的数量明显增多,同时对小鼠血清中HBsAg及HBVDNA水平有明显的抑制作用.肝组织HBsAg免疫组织化学蛋白平均吸光度分析显示,50μg和100μgPTD-HBcAg融合蛋白组中平均吸光度值与空白组和50μgHBcAg组相比明显降低(127.77±4.92,117.71±5.18vs156.84±4.94,138.70±5.92,均P<0.05),且组间比较差异有统计学意义.结论:PTD-HBcAg融合蛋白免疫HBV转基因小鼠后能增加特异性CTL数量,显著降低血清中HBsAg及HBVDNA水平,同时抑制肝脏中HBsAg的表达,在HBV免疫治疗中具有抗病毒作用.

AIM： To investigate the effect of protein transduction domain-hepatitis B virus core antigen （PTD-HBcAg） fusion protein-induced specific cytotoxic T lymphocyte （CTL） response on hepatitis B virus （HBV） replication in HBVtransgenic mice. METHODS： Twenty HBV-transgenic mice were randomly divided into two groups： mice subcutaneously immunized with PTD-HBcAg fusion protein （PTD-HbcAg group） and those immunizedwith HBcAg （HBcAg group）. The immunizations were given once a week for three weeks. Cytokine expression in splenocytes was analyzed by flow cytometry. Serum HBsAg and HBV DNA levels were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. The expression of HBsAg in hepatic tissue was detected by immunohistochemistry. RESULTS： Following immunization with PTD- HBcAg fusion protein, the proportions of CTLs in the spleen and inflammatory cells in hepatic tissue of HBV transgenic mice were upregulated remarkably. Serum HBsAg and HBV DNA levels in the PTD-HbcAg group were significantly lower than those in the HBcAg group. The mean absorbance values of HBsAg staining in hepatic tissue in the PTD-HBcAg group （50μg： 127.77 ± 4.92; 100μg： 117.71 ±5.18） were significantly lower than those in the blank control group （156.84 ±4.94） and HBcAg group （50μg： 138.70 ± 5.92）（all P 〈 0.05）. CONCLUSION： Immunization with PTD- HBcAg fusion protein significantly increases the number of specific CTLs in the spleen, decreases serum HBsAg and HBV DNA levels, and down- regulates HbsAg expression in hepatic tissue in HBV-transgenic mice, suggesting that PTD- HBcAg fusion protein has anti-HBV activity.