基于假病毒的人乳头瘤病毒小鼠感染模型的建立及HPV16VLP疫苗保护性评价  

Establishment of human papillomavirus pseudovirion infection model in mouse for potency evaluation of HPV16 VLP Vaccine

作　　者：王大宁[1] 张丽[2] 刘亚静[1] 魏旻希 夏宁邵[1,4] 李少伟[1,4] 

机构地区：[1]厦门大学公共卫生学院/分子疫苗学与分子诊断学国家重点实验室,福建厦门361102 [2]厦门大学医学院,福建厦门361102 [3]厦门万泰沧海生物技术有限公司,福建厦门361000 [4]厦门大学生命科学学院/国家传染病诊断试剂与疫苗工程技术研究中心,福建厦门361102

出　　处：《中国生化药物杂志》2015年第11期5-10,4,共6页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家自然科学基金(81172885)

摘　　要：目的建立基于假病毒的人乳头瘤病毒(human papillomavirus,HPV)小鼠感染模型并应用于HPV16 VLP疫苗的保护性评价。方法利用293FT细胞制备携带Luc报告基因的HPV16假病毒,纯化后检测HPV16假病毒滴度和性质;利用醋酸甲羟孕酮和β-雌二醇处理雌性BAL B/c小鼠,然后用壬苯醇醚-9(N-9)化学损伤BALB/c小鼠阴道,6 h后用HPV16假病毒感染小鼠阴道,48 h后使用活体检测仪检测小鼠阴道中Luc报告基因的表达情况;最后,利用该感染模型评价HPV16 VLP疫苗的保护能力。结果获得了含有Luc报告基因的HPV16假病毒,建立了HPV假病毒纯化方法;Western blot结果显示HPV16假病毒中含有L1和L2蛋白;建立了HPV假病毒感染小鼠模型,并通过不同剂量的假病毒感染实验表明,该感染模型所需的最低假病毒剂量是1.7×104TRLU;利用该模型获知HPV16 VLP疫苗具有较强的抵抗HPV16假病毒感染的能力,当中和抗体滴度为256时即可阻碍HPV假病毒感染。结论本研究成功地建立了HPV假病毒小鼠感染模型,并运用该模型评价了HPV16 VLP疫苗的保护作用,为HPV中和抗体研究和候选疫苗的免疫保护评价提供了有效工具。Objective To establish a mouse model of genital human papillomavirus（ HPV） pseudovirion（ Ps V） transmission and evaluate the protective potency of HPV16 VLP vaccine. Methods HPV16 Ps V with the encapsidated luciferase expressing plasmid Luc were generated from 293 FT cells and purified by size-exclusion chromatography. The endpoint titers of HPV16 Ps V-Luc were determined on 293 FT cells,denoted as TRLU / m L. For in vivo genital challenge,mice were synchronized in a diestrus-like status by a subcutaneous injection with 0. 1 μg β-estradiol and then with 3mg Depo Provera after 24 hours. Six hours prior to HPV16 Ps V-Luc challenge,deeply anesthetized mice were intravaginally pretreated with 50 μL of 4%nonoxynol-9（ N-9）. HPV16 Ps V-Luc was thoroughly mixed with 20 μL solution containing 4% carboxymethylcellulose（ CMC） and intravaginally instilled using a positive-displacement pipette. Forty-eight hours after Ps V-Luc challenge,mice were anesthetized and D luciferin was intravaginally instilled. After 3 minites,bioluminescence was measured with a cryogenically cooled Xenogen IVIS camera system. Then,the murine genital challenge model was used to determine the potency that HPV16 VLP vaccine is efficient at preventing HPV infection. Results HPV16 Ps V-Luc was generated and purified from 293 FT cells. HPV16 Ps V-Luc was verified to containe L1 and L2 protein by Western blot. HPV 16 Ps V-Luc successfully infected vaginal epithelial cells of mouse and the murine genital challenge model was established. To achieve consistent bioluminescence,the minimal dose of HPV16 Ps V-Luc was 1. 7 × 104 TRLU. The protective potency of HPV16 VLP vaccine was shown using this murine model. Our data showed that immune serum with over neutralizing antibody titer of 256-fold was sufficient to confer protection against HPV Ps V genital infection. Conclusion The murine genital challenge model of HPV16 was successfully established,and the model is used to evaluate the potency of HPV16 VLP vaccine against in vivo genital HPV1

关 键 词：人乳头瘤病毒 假病毒 小鼠感染模型 中和保护 

分 类 号：R373[医药卫生—病原生物学]