乙脑病毒prME与E蛋白编码基因重组构建的DNA免疫试验研究

DNA immunization by recombinants encoding prME and E proteins derived from Japanese encephalitis virus

目的 研究乙脑病毒prME、E蛋白的体外表达特点 ,比较不同重组质粒所进行的DNA免疫效率。方法 分别将乙脑病毒 (JaGAr 0 1株 )prME、E蛋白编码基因 (2 0 0 1bp、15 0 0bp)构建于融合FLAG标记基因的真核表达载体pcDNA(FLAG) 3上 (pJME、pJE) ,脂质体法将二重组质粒转染于HepG2及COS 1细胞系 ;采用不同抗体系统 (anti FLAG、anti E) ,经Westernblot法检测乙脑病毒prME[相对分子质量 (Mr)约 72× 10 3]与E(Mr 约 5 4× 10 3)蛋白表达水平 ;将质粒肌注BALB c鼠 ,二次免疫后 3周 ,腹腔注射乙脑病毒 (10 5PFU 10 0 μl)作为病毒攻击并观察 2 1d。 80 %空斑减少中和实验法检测中和抗体滴度变化。结果 anti FLAG可检测到由质粒pJME、pJE编码的相应蛋白产物在转染细胞内表达 ,同时在pJME转染的细胞内检测到一个新的 11× 10 3的低Mr 蛋白。anti E仅在pJME转染的细胞内检测到E蛋白。肌注pJME可产生高水平中和抗体滴度以及诱导有效的保护性免疫 ,效果等同于普通灭活JE疫苗 ,但优于pJE。结论 pJME的体外表达水平优于pJE。DNA免疫实验结果与prME。

Objective To study the expression characteristic of proteins from prM to E derived from JEV and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2?001bp) and E ( 1?500bp ) genes respectively. Methods Two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS 1 cells by lipsome fusion. The expression feature of FLAG prME (about 72kD) and FLAG E (about 54kD) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti FLAG and anti E). BALB/c mice were immunized with 100μg of two kinds of recombinants by intramuscular (im) injection respectively. JEV JaGAr 01 strains (10 5PFU/100μl) were given to BALB/c mice by intraperioneal (ip) injection 3 weeks after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. Results The expression of proteins associated with pJME and pJE was determined in transfected cells with anti FLAG and then a new protein of 11kD was detected in HepG2 and COS 1 cells transfected with pJME. Only E (53kD) protein was identified as transfected with pJME using anti E. Higher level of neutralization antibodies and efficacy of protective immunity were induced with pJME immunization, and were similar to those induced with inactivated JE vaccine, but were better than those induced with pJE. Conclusion The expression level from prM to E proteins of JEV is different in vitro . The expression efficiency of pJME in vitro was better than that of pJE. FLAG prME protein expressed by pJME could be cleaved by peptidase from host factors. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro . [