汉逊酵母重组表达的人乳头瘤病毒58型类病毒颗粒的免疫效果  

Evaluation of immune effect of virus-like particles of Hansenula polymorpha-based recombinant human papillomavirus type 58

作　　者：靳玉琴[1] 侯俊伟[1] 张靖[1] 陈实[1] 李启明[1] 

机构地区：[1]北京生物制品研究所有限责任公司,北京101111

出　　处：《中国生物制品学杂志》2013年第12期1697-1700,1704,共5页Chinese Journal of Biologicals

基　　金：863计划重大项目(2012AA02A401)

摘　　要：目的评价基于汉逊酵母系统的重组人乳头瘤病毒(human papillomavirus,HPV)58型L1蛋白类病毒颗粒(virus-like particles,VLP)的免疫效果。方法将HPV58 L1重组汉逊酵母工程菌转种于3.7 L发酵罐中,经甲醇诱导15 h,收集菌体,高压均质破碎菌体后,采用亲和层析法纯化重组HPV58 L1蛋白,纯化后的HPV58 L1经重构后,经透射电镜观察VLP形态,动态光散射仪分析比较重构与未重构VLP粒径大小及分布情况。将HPV58 L1蛋白(1μg/ml)及HPV58 L1蛋白+佐剂(蛋白含量1μg/ml,铝含量0.41 mg/ml)分别免疫BALB/c小鼠,免疫后4周采血分离血清,假病毒中和试验比较铝佐剂吸附与未吸附免疫效果;将HPV58 L1蛋白+佐剂(蛋白含量1μg/ml,铝含量0.41 mg/ml)免疫5组BALB/c小鼠,每组免疫时间均为0、1、3、5、7周,于初次免疫后第1、2、4、6、8周采血,分离血清,假病毒中和试验检测HPV58 L1免疫效果。结果纯化后的重组HPV58 L1蛋白纯度约90%,可与小鼠抗HPV58 L1单克隆抗体发生特异性结合,在相对分子质量约56 000处,可见特异性条带。重构的HPV58 L1VLP直径约为50 nm,界限清晰,颗粒均一度较高,更接近天然病毒的颗粒形态;流体力学直径与天然病毒颗粒更接近,颗粒大小分布更均一,无较小半径的单体或五聚体。HPV58 L1蛋白+佐剂免疫小鼠血清中和抗体滴度明显高于HPV58 L1蛋白免疫组,差异有统计学意义(P<0.05);5组小鼠血清中和抗体滴度在初次免疫后1周即开始升高,第6周达到最高,重构的重组HPV58 L1蛋白可诱导小鼠产生较高水平的中和抗体。结论应用含铝佐剂的HPV58 L1 VLP免疫小鼠,具有较好的免疫学活性,可作为多价HPV疫苗的组分抗原。Objective To evaluate the immune effect of virus-like particles （VLPs） of L1 protein of Hansenula polymorph-based human papillomavirus type 58 （HPV58）. Methods The recombinant H. polymorph for expression of HPV58 L1 was transferred to 3.7 L fermenter and induced with methanol for 15 h, then collected and broken by high pressure homogenization, from which recombinant HPV58 L1 protein was purified by affinity chromatography, reconstituted, and observed for morphology of VLPs by transmission electron microscopy. The sizes and their distributions of before and after reconstitution were compared by dynamic light scattering analyzer. BALB / c mice divided into two groups and immunized with 1 p.g/ml HPV58 L1 and 1 μg/ml HPV58 L1 ＋ 0. 41 mg/ml aluminium adjuvant respectively, of which sera were collected 4 weeks later to compare the immune effect by pseudovirus neutralization test. Five groups of mice, were immunized with 1 μg/ml HPV58 L1 ＋ 0. 41 mg/ml aluminium adjuvant at weeks 0, 1, 3, 5 and 7, of which sera were coUected at weeks 1, 2, 4, 6 and 8 after the first immunization to evaluate the immune effect by pseudoviurs neutralization test. Results The purified recombinant HPV58 L1 reached a purity of about 90% and showed specific binding to mouse monoclonal antibody against HPV58 L1, which showed a specific band with a relative molecular mass of about 56 000 on SDS-PAGE profile. The VLPs of reconstituted HPV58 L1 were at a mean diameter of about 50 nm, with clear border and high homogeneity, of which the morphology was similar to that of natural virus. Compared with those

关 键 词：汉逊酵母 人乳头瘤病毒58型 类病毒颗粒 免疫效果 

分 类 号：R373.9[医药卫生—病原生物学] R392.33[医药卫生—基础医学]