乙型脑炎病毒prME、E蛋白基因表达与DNA免疫效率对比研究

Comparison of gene expression and DNA immunization efficacy encoding prME and E proteins derived from Japanese encephalitis virus

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 expression characterization of prME, E proteins derived from Japanese encephalitis virus （JEV） and DNA immunization efficacy induced by different routes of inoculation. Methods Both of recombinants containing JEV prME and E genes, named after pJME and pJE respectively, were transfected into HepG2, COS-1 and KN73 cells by lipsome fusion. The expression feature of protein encoded by pJME and pJE in transfected cells, the relationship between plasmid amount for transfection and protein expression, and the antibody from BALB/c mouse sera post-DNA immunization were analyzed by Western blot. BALB/c mice were vaccinated with 100μg of pJME and pJE by intramuscular （ira.）injection, and 3μg of pJME and pJE by gene gun injection respectively. JEV JaGAr-01 strains （10^5 PFU/100μl） were given to BALB/c mice by intraperioneal （ip） injection for virus challenge. 80% plaque reduction neutralization test （PRNT80） was performed to titrate neutralization antibody. Results About 72 kD of proteins and 11 kD of protein were determined in HepG2 and COS-1 cells transfected with pJME. About 54 kD of proteins were determined in HepG2, COS-1 and KN73 cells transfected with pJE. The expression level of proteins in HepG2 cells transfected with pJME from higher to lower followed as 10μg/well〉5μg/well〉3μg/well. All BALB/c mice immunized with pJME by im. and gene gun in3ectton, ano 3m mac for 21 days. BALB/c mice immunized with pJE by im. injection and gene gun injection partially survived for 21 days. Titres of neutralization antibody produced with pJME were higher than pJE. Protective immunity and titre of neutralization antibody produced by im injection was the same as gene gun injection（im/gene gun injection~ 1 ： 320/1 ~ 320） at day 21. The antibody from BALB/c mice sera after twice pJME immunization only reacts with JEV-E protein. Conclusions Expression efficacy of proteins encoded by pJME and pJE in transfected cells is different. Expression level of related proteins was dependant on recombinant amount for transfection in a certain degree. Immunity effect induced with pJME was higher than pJE. The efficacy of DNA immunization produced by gene gun injection was higher than ira. injection. Titres of neutralization antibody induced by DNA immunization were correlated to efficacy of protective immunity. Neutralization antibody from BALB/c mice sera produced by pJME immunization contained anti-E antibody against JEV-E protein.