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目的探讨丙型肝炎病毒(HCV)包膜E2蛋白DNA疫苗诱导小鼠产生中和抗体的可行性。方法构建截除疏水性羧基末端的HCV包膜蛋白表达质粒pCI-1b661以及同时截除疏水性羧基末端和高变区1(HVR1)的表达质粒pCI-1b661Δ,转染293T细胞,以Western blot和ELISA检测细胞内和培养上清中的HCVE2蛋白,将两种表达质粒及空载体分别肌注免疫BALB/c小鼠,以ELISA检测小鼠血清中的HVR1抗体,以HCV假病毒颗粒(HCVpp)分析小鼠血清的中和活性。结果 2种表达质粒均能表达分泌性截短型E2蛋白。pCI-1b661免疫的8只小鼠血清中均可检测到HVR1抗体,而pCI-1b661Δ免疫血清中未检测到HVR1抗体。pCI-1b661和pCI-1b661Δ免疫血清对HCVpp的中和率分别为(78.5±13.8)%和(38.7±6.5)%,差异有显著性(P<0.01)。pCI-1b661免疫组小鼠血清的中和率与HVR1抗体水平呈正相关(r=0.967,P<0.01)。结论表达截短型E2蛋白的DNA疫苗能诱导产生HCV中和抗体,其主要成员为HVR1抗体。
Objective To investigate the feasibility of producing neutralizing antibodies in mice induced by HCV envelope glycoprotein E2 DNA vaccine. Methods The recombinant plasmid pCI-1b661 was constructed by cloning the hydrophobic carboxy-terminal HCV envelope protein (pCI-1b661) and pCI-1b661Δ. The recombinant plasmid pCI-1b661Δ was transfected into 293T cells by Western blot and Western Blot ELISA was used to detect HCVE2 protein in the cells and culture supernatants. BALB / c mice were immunized with the two expression plasmids and empty vector respectively, and the HVR1 antibody in serum was detected by ELISA. HCV pseudovirions (HCVpp) The mouse serum neutralization activity was analyzed. Results Both expression plasmids could express secreted truncated E2 protein. The HVR1 antibody was detected in the sera of 8 mice immunized with pCI-1b661, while no HVR1 antibody was detected in the pCI-1b661Δ immune serum. The neutralization rates of pCI-1b661 and pCI-1b661Δ immune serum to HCVpp were (78.5 ± 13.8)% and (38.7 ± 6.5)%, respectively, with significant difference (P <0.01). The neutralization rate of pCI-1b661 immunized mice was positively correlated with HVR1 antibody level (r = 0.967, P <0.01). Conclusion The DNA vaccine expressing the truncated E2 protein can induce the production of HCV neutralizing antibody. The main member of the vaccine is HVR1 antibody.