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目的研究乙型脑炎病毒prME、E蛋白表达特点,比较不同接种途径所致DNA免疫效率。方法脂质体法将质粒(pJME、pJE)转染于HepG2、COS-1及KN73细胞;免疫印迹法分析质粒表达及与转染剂量关系,鉴定DNA免疫鼠血清抗体性质;肌内(100μg/次)与基因枪(3μg/次)注射法将质粒免疫BALB/c鼠,腹腔注射乙型脑炎病毒(105PFU/100μl)进行病毒攻击。80%空斑减少中和试验法检测中和抗体滴度。结果pJME转染的细胞内可检测到相对分子质量约为72×103与11×103两种蛋白,pJE转染的细胞内检测到相对分子质量约为54×103蛋白。蛋白表达程度由高至低顺序为10μg/孔>5μg/孔>3μg/孔。pJME与JE灭活疫苗免疫的BALB/c鼠在病毒攻击后21 d全部存活,pJE免疫鼠部分存活。pJME所致中和抗体滴度高于pJE。肌内注射组所致的最终中和抗体滴度等同于基因枪注射组。pJME免疫鼠产生血清抗体仅与JEV E蛋白发生反应。结论pJME与pJE在不同转染细胞内表达效率不同;转染剂量和蛋白表达呈一定剂量依赖性;pJME免疫效果优于pJE,基因枪注射所致免疫效率高于肌内注射;DNA免疫所致的中和抗体效价水平与保护性免疫效果呈一致性;DNA免疫鼠血清中和抗体含针对JEV E蛋白的抗JEV-E抗体。
Objective To study the expression of prME and E protein of Japanese encephalitis virus (JEV), and to compare the DNA immunization efficiency caused by different routes of vaccination. Methods The plasmids (pJME, pJE) were transfected into HepG2, COS-1 and KN73 cells by lipofectamine. The expression of plasmids was analyzed by immunoblotting, BALB / c mice were immunized with the plasmid (3μg / time) by injection and the encephalitis virus (105PFU / 100μl) was intraperitoneally injected for virus challenge. Neutralizing antibody titers were detected by the 80% plaque reduction and neutralization assay. Results The relative molecular mass of pJME-transfected cells was about 72 × 103 and 11 × 103, respectively. The relative molecular mass of pJME-transfected cells was 54 × 103. Protein expression levels from high to low were 10 μg / well> 5 μg / well> 3 μg / well. BALB / c mice immunized with pJME and JE inactivated vaccine all survived 21 d after virus challenge, and part of pJE immunized mice survived. The pJME-induced neutralizing antibody titers were higher than pJE. The final neutralizing antibody titer resulting from the intramuscular injection group was equivalent to the gene gun injection group. The pJME-immunized mice produce serum antibodies that react only with the JEV E protein. CONCLUSION: The expression efficiency of pJME and pJE in different transfected cells is different. The transfection dose and protein expression are dose-dependent. The pJME is better than pJE in immunogenicity. The immunization efficiency is higher than that of intramuscular injection by gene gun injection. Of neutralizing antibody titers were consistent with the protective immune effect. DNA immunized rat serum-neutralizing antibody contained anti-JEV-E antibody against JEV E protein.