手足口病CA16型VP1亚单位疫苗的构建制备及免疫原性初步分析  

Preparation of the Coxsackievirus A16 VPI subunit vaccine and detection of its immunogenicity

作　　者：李晓楠[1,2] 罗德炎[2] 赵忠鹏[2] 段越强[2] 李培锋[1] 王希良[2] 

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验,北京100071

出　　处：《中华微生物学和免疫学杂志》2010年第3期250-255,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的制备CA16型VP1蛋白疫苗并进行免疫原性初步分析，为手足VI病CA16疫苗的深入研究提供资料。方法利用RT—PCR技术获得CA16VP1基因，克隆到载体pFastBacHTA，与BacmidDNA重组，转染Si9昆虫细胞，重组CA16VP1蛋白与AI（OH），佐剂混合制备重组CA16VP1蛋白疫苗，腹腔免疫BALB／c小鼠，2次免疫后进行免疫效果初步评价。结果用间接免疫荧光、SDS—PAGE和Westernblot法，证明重组CA16VP1蛋白在昆虫细胞St9中得到表达，用ELISA和微量中和法检测到免疫小鼠血清有特异IgG和中和抗体产生，最佳免疫抗原剂量为20μg，其特异IgG效价为1：1600，中和抗体效价为1：250；通过淋巴细胞增殖试验和细胞因子测定，证明诱导T细胞应答，诱发Th1／Th2型免疫应答。结论CA16VP1基因克隆成功，并在Sf9昆虫细胞中获得表达，构建的重组CA16VP1亚单位疫苗具有诱导特异性细胞免疫和体液免疫应答的能力，为今后研制手足口病CA16疫苗奠定了基础。Objective To prepare VP1 protein vaccine of Coxsackievirus A16 （CA16） and evaluate immunogenicity the subunit vaccines of Coxsackievirus （ VP1 ）, and to establish foundation for studying CA16 vaccine. Methods CA16 VP1 was amplified by RT-PCR and cloned into pFastBac HT A plasmid, recombinated with Baemid DNA by transposition reaction and then transfected St9 cell, mixed with adjuvant AI（OH）3. After immunization BALB/c mice, evaluating immune effectiveness after booster injections 2 weeks. Results The expressed protein was analyzed by SDS-PAGE and Western blot, mice immunized with CA16 （ VP1 ） both induced specific IgG antibody and neutralization antibody. The best immunization antigen was 20 μg, IgG antibody was 1： 1600, neutralization antibody was 1： 250, typical Thl/Th2 immune response was determined by lymphocyte proliferation assay and cytokine analysis. Conclusion The CA16 VP1 gene was cloned successfully and expressed in St9 insect cells, CA16 VP1 protein vaccine induced both humoral and cellular immune response, to lay solid foundation for further study on CA16 vaccine.

关 键 词：CA16病毒 Bac—to—Bac表达系统 重组VP1蛋白 免疫原性 

分 类 号：R392[医药卫生—免疫学]