丙型肝炎病毒E2蛋白DNA疫苗诱导小鼠产生中和抗体的研究

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。

Study of neutralization antibodie induced by DNA vaccine of HCV envelope protein 2 in mice

Objective To explore the feasibility of induction of neutralization antibodies against hepatitis C virus(HCV) infection by HCV envelope 2 protein(E2) DNA vaccines immunization.Methods Two kinds of expression plasmids of HCV envelope 2 protein,plasmid pCI-1b661 encoding hydrophobic carboxyl terminal truncated E2 and pCI-1b661Δ encoding E2 with deletion of hypervariable region 1(HVR1) and carboxyl terminal,were constructed and respectively transfeted 293T cells,and truncated E2 protein in whole cell lysate and supernatant of 293T cells were analyzed by Western blot.After BALB/c mouse were intramuscularly immunized by the plasmids,sera antibodies against HVR1 were detected by ELISA and the neutralization activity of the antibodies were assayed with HCV pseudotype particle(HCVpp).Results Both plasmids could express secretary truncated E2 protein.All the mice immunized with plasmid pCI-1b661 produced HVR1 antibodies,while no HVR1 antibodies were detected in pCI-1b661Δ immunized mice.The sera neutralization percentages against HCVpp in pCI-1b661 and pCI-1b661Δ immunized mice were(78.5±13.8)% and(38.7±6.5)%,respectively(P<0.01).Sera neutralization activity against HCVpp was positive correlated with the level of HVR1 antibodies in pCI-1b661 immunized mice(r=0.967,P<0.01).Conclusion DNA vaccines expressing truncated E2 protein could induce neutralization antibodies against HCV,and neutralization antibodies mainly was consisted of the antibodies against HVR1.