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目的探讨HCV包膜E2蛋白DNA疫苗诱导小鼠产生中和抗体的可行性。方法分别构建HCV E2蛋白全长表达质粒pcDNA3.1-1a746、截除疏水性羧基末端的表达质粒pcDNA3.1-1a661以及同时截除疏水性羧基末端和高变区1(HVR1)的表达质粒pcDNA3.1-1a661Δ,转染293T细胞,以Western blot和ELISA法检测细胞内和培养上清中的HCV E2蛋白,将3种表达质粒以及空载体分别肌注免疫BALB/c小鼠,检测小鼠血清的E2及HVR1抗体,以HCV假病毒模型分析小鼠血清的中和活性。结果3种E2表达质粒均能有效表达HCV E2蛋白,其中pcDNA3.1-1a746质粒的表达产物不能分泌,而pcDNA3.1-1a661和pcDNA3.1-1a661Δ均能分泌表达E2蛋白。分泌表达E2蛋白可显著增强小鼠的抗体应答,pcDNA3.1-1a661免疫血清对HCV假病毒颗粒(HCVpp)的中和活性明显高于pcDNA3.1-1a661Δ免疫血清。pcDNA3.1-1a661免疫血清中的HVR1抗体量仅占总E2抗体的一小部分,却是中和抗体的重要成分。结论表达E2蛋白的DNA疫苗能有效诱导HCV中和抗体的产生,HVR1不仅是重要的中和抗体表位,而且能增强E2蛋白其他抗原表位的抗体应答。
Objective To investigate the feasibility of using HCV envelope E2 protein DNA vaccine to induce neutralizing antibodies in mice. Methods The full-length HCV E2 expression plasmid pcDNA3.1-1a746, the expression plasmid pcDNA3.1-1a661 excluding the hydrophobic carboxyl terminus and the expression plasmid pcDNA3 harboring both the carboxy terminal and hypervariable region 1 (HVR1) .1-1a661Δ were transfected into 293T cells. HCV E2 protein in the cells and culture supernatants were detected by Western blot and ELISA. BALB / c mice were immunized with three expression plasmids and empty vector, respectively. Serum E2 and HVR1 antibodies were analyzed for the neutralizing activity of mouse sera using the HCV pseudovirus model. Results All of the E2 expression plasmids could efficiently express HCV E2 protein, of which the expression product of pcDNA3.1-1a746 plasmid could not be secreted, while both pcDNA3.1-1a661 and pcDNA3.1-1a661Δ secreted E2 protein. Secretion of E2 protein can significantly enhance the antibody response in mice, pcDNA3.1-1a661 immune serum of HCV pseudovirus particles (HCVpp) was significantly higher than the neutralizing activity of pcDNA3.1-1a661Δ immune serum. The amount of HVR1 antibody in the pcDNA3.1-1a661 immune serum is only a fraction of the total E2 antibody but is an important component of the neutralizing antibody. Conclusion The DNA vaccine expressing E2 protein can effectively induce the production of HCV neutralizing antibody. HVR1 is not only an important neutralizing antibody epitope, but also enhances the antibody response of other epitopes of E2 protein.