热休克蛋白65增强小鼠对HBV DNA疫苗免疫反应的研究

Enhancement of immune responses to hepatitis B DNA vaccine by heat shock protein 65 in mice

目的：观察热休克蛋白65的真核表达载体（pHSP65）对HBVDNA疫苗诱导BALB／c小鼠（H-2^d）免疫应答的调节作用。方法：肌内注射空栽体pcDNA3，HBVDNA疫苗加HSP65佐剂（pHBVS2S＋pHSP65）或不加佐剂（pHBVS2S）；ELISA法测定血清抗HBs抗体；ELISPOT检测分泌IFN-γ的脾淋巴细胞；4h^51Cr释放法检测小鼠脾细胞CTLs活性。结果：HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度虽高于不加佐剂组，但无显著性差异；其IgG1／IgG2a的比例不同于多肽免疫组，二者分别为0．395与10。佐剂组小鼠分泌IFN-γ的脾淋巴细胞量是不加佐剂组的2—3倍。CTL与细胞杀伤活性（E：T=100）佐剂组与不加佐剂组分别为：（53．68±7．5）％、（42．81±7．7）％，差异显著（P〈0．05）。结论：HBVDNA疫苗具有较强的免疫原性，能够诱导机体产生特异性的抗体及CTLs反应；HSP65佐剂能够有效提高小鼠对DNA疫苗的细胞免疫应答，有望成为DNA疫苗的免疫佐剂。

Objective：To observe the effect of eukaryotic expression vector pHSP65 coding heat shock protein 65 on immune responses induced by DNA immunization of HBV surface antigen （pHBVS2S） in BABL/c （H-2^d） mice . Methods： The immunization was performed by intra-muscular injection of empty vector pcDNA3 ,HBV DNA vaccine （pHBVS2S） with or without pHSP65 adjuvant plasmid. Anti-HBs in serum was detected by ELISA and HBsAg specific cytotoxic T lymphocytes （CTLs） activity was measured by ^51 Chromium Release Assay. The number and affinity of HBs peptide antigen-specific IFN-γ secreting T cells were detected by ELISPOT, Results. The CTLs activity of mice in pHBVS2S ＋ pHSP65 group was significanly higher （P 〈 0. 05） than that in mice intramuscular injected pHBVS2S DNA vaccine group. The anti-HBs titer of mouse serum in pHBVS2S ＋ pHSP65 group was higher than that in mice intramuscularly injected pHBVS2S DNA vaccine group, The ratio of IgG1/IgG2a was different from the group immunized with peptide and was 0. 395 and 10 in the pHBVS2S ＋ pHSP65 group and the rHBsAg group respectively. The number of HBs peptide antigen-specific IFN-γ-secrering T cells in pHSP65 adjuvant group was 2 -3 times higher than that in pHBVS2S DNA vaccine group. Conduslon：The DNA vaccine of HBV （pHBVS2S） has strong immunogenecity and the HSP65 adjuvant can enhance the immune response to henatitis B DNA vaccine in mice, Thus the DHSP65 may be a Promising adjuvant for DNA vaccine.