柯萨奇病毒B3 VP1蛋白、rAd/VP1和pcDNA3/VP1的免疫效果比较  

Comparison of the immune effects of Coxsackievirus B3 VP1 protein, rAd/VP1 and pcDNA3/VP1 in mice

作　　者：蓝佳明[1] 高志云[1] 李嘉[1] 金玉怀[1] 温婵[2] 李伟[3] 闫立景 刘贵霞[1] 谢立新[1] 王永祥[1] 

机构地区：[1]河北医科大学病原生物学教研室,石家庄050017 [2]河北省儿童医院感染控制处 [3]河北大学附属医院检验科 [4]江南大学校医院

出　　处：《中华微生物学和免疫学杂志》2011年第1期25-29,共5页Chinese Journal of Microbiology and Immunology

基　　金：河北省科学技术研究与发展计划（08276101D-93）;河北省医学科学研究重点课题（08258,20090054）

摘　　要：目的 观察柯萨奇病毒B3（Coxsackievirus B3,CVB3）衣壳蛋白VP1、表达VP1蛋白的重组腺病毒rAd/VP1和重组质粒pcDNA3/VP1的免疫效果.方法 用原核细胞表达VP1蛋白并纯化、扩增重组腺病毒rAd/VP1,扩增并提取真核表达质粒pcDNA3/VP1.BALB/c小鼠随机分为4组,每组18只,分别在股四头肌注射VP1蛋白、rAd/VP1、pcDNA3/VP1和PBS.VP1蛋白组和pcDNA3/VP1组免疫3次,间隔3周;rAd/VP1组免疫2次,间隔2周.VP1蛋白、pcDNA3/VP1和rAd/VP1每次每只注射剂量分别为50μg、100μg和1.2×107PFU.用ELISA法和微量中和试验法检测各次免疫后血清CVB3特异性IgG抗体和中和抗体滴度;末次免疫后3周,CCK-8法检测脾脏淋巴细胞的CTL杀伤活性;用致死量CVB3攻击小鼠后,检测血中病毒滴度并观察动物的存活情况.结果 VP1蛋白组血清特异性IgG抗体和中和抗体滴度明显高于其他实验组（P＜0.05）,而脾脏淋巴细胞CTL杀伤活性低于rAd/VP1组（P＜0.05）;致死量病毒攻击后,VP1蛋白组血中病毒滴度低于pcDNA3/VP1和rAd/VP1组（P＜0.05）,生存率明显高于这两组（P＜0.05）.结论 VP1蛋白疫苗能诱导较高水平的体液免疫应答,对动物有明显的免疫保护作用,免疫效果优于质粒pcDNA3/VP1和重组腺病毒rAd/VP1.Objective To compare the immune effects of Coxsackievirus B3 （CVB3） capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte（CTL） killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose（ CCID50 ） assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups（ P 〈0.05 ）. While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group（ P 〈0. 05）. Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups （ P 〈 0.05 ）, while survival rate was significantly higher than

关 键 词：柯萨奇病毒B3 腺病毒载体疫苗 蛋白疫苗 DNA疫苗 免疫应答 

分 类 号：R3[医药卫生—基础医学]