摘要
目的:观察不同免疫途径和佐剂类型对柯萨奇病毒B组3型(Coxsackievirus group B type 3,CVB3)衣壳蛋白VP1免疫效果的影响。方法:将原核表达质粒pET-his/VP1转入E.coli BL21(DE3)pLysS中,用异丙基-1-硫代-β呋喃半乳糖苷(IPTG)诱导CVB3VP1蛋白的表达并进行纯化。首先采用不同的免疫途径(皮下,腹腔,肌肉)用VP1蛋白免疫小鼠,每组12只。然后另取小鼠分为PBS组和不同佐剂组(氢氧化铝、弗氏佐剂、Montanide ISA720),每组18只,采用肌肉注射途径免疫。每次每只小鼠注射50μg,共免疫3次,间隔3周。用ELISA和微量中和试验检测血清特异性IgG抗体和中和抗体。用CCK-8法检测淋巴细胞增殖活性和CTL杀伤活性。用致死量的CVB3攻击后,检测血中病毒的滴度并观察小鼠的存活状况。结果:在大肠杆菌中成功表达CVB3VP1蛋白。三种免疫途径比较,肌肉注射组血清中和抗体和特异性IgG抗体的水平明显高于其他组(P<0.01)。采用肌肉注射免疫时,弗氏佐剂组Montanide ISA 720佐剂组的体液免疫和细胞免疫应答的水平明显高于氢氧化铝组(P<0.05);但血中病毒的滴度低于氢氧化铝组(P<0.05)。弗氏佐剂组小鼠的生存率好于氢氧化铝组(P<0.05)。结论:采用肌肉注射途径,并联合弗氏佐剂或Montanide ISA 720佐剂可以使CVB3VP1免疫获得较好的免疫效果。
AIM： To explore the effects of inoculation route and adjuvant type on the immunizing potency of coxsackievrus B type 3 （CVB3） VP1 protein. METHODS： The recombinant plasmid pET-His/VP1 expressed CVB3 VP1 was transformed into E.coli BL21（DE3） pLysS and induced to express VP1 protein by IPTG, and verified by Western blot analysis. The fusion VP1 protein was purified with Ni affinity chromatography. Firstly, BALB/c mice were administered via different inoculation route（subcutaneous, intraperitoneal, intramuscular）, with twelves mice in each group. Secondly, combined with various adjuvants （Alum, Freund’s adjuvant, Montanide ISA720）, with eighteen mice in each group. The mice were immunized three times at a three week interval with 50 μg of VP1 protein. The titers of sera IgG and neutralizing antibody were detected by ELISA and neutralization assay. Cell mediated immune response was tested by the lymphocytes proliferation activity and specific CTL cytotoxic activity. The mice were challenged with lethal dose of CVB3, the titers of the sera virus were titrated. Furthermore, the survival rates of mice were observed. RESULTS： The VP1 protein was expressed in E.coli successfully and the fusion protein was purified. In different inoculation route, the titers of neutralizing antibody and specific IgG in intramuscular injection group was much higer than other groups （P〈0.01）. VP1 protein in combination with Montanide ISA720 and Freund’s adjuvant elicit higher titer antibodies and cell mediated immune response, and the virus titers in blood were lower in comparison to Alum adjuvant group （P〈0.05）.The survival rate of Freund’s adjuvant group was better than adjuvant AL（OH）3 group （P〈0.05）. CONCLUSION： The VP1 protein combined with ISA720 and Freund’s adjuvant given by intramuscular injection may induce an improved immune responses and the better survival rates of the mice after virus challenge.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2011年第10期1086-1089,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
河北省科学技术研究与发展计划资助项目(08276101D-93)
河北省医学科学研究重点课题资助项目(20090049)
