人乳头瘤病毒31和33型L1蛋白类病毒颗粒的制备及其免疫原性

发布时间：2018-10-21 08:28

【摘要】：目的采用汉逊酵母系统表达人乳头瘤病毒(human papillomavirus,HPV)31和33型L1蛋白类病毒颗粒(virus-like particles,VLPs),纯化后检测其免疫原性。方法将表达HPV31和HPV33 L1蛋白的重组汉逊酵母工程菌经高密度发酵,收集菌体,进行高压均质破碎后,采用亲和层析法纯化HPV31和HPV33 L1 VLPs,SDS-PAGE和Western blot鉴定重组蛋白,透射电镜观察VLPs形态,动态光散射仪分析VLPs粒径大小及分布情况。将BALB/c小鼠随机分为3组:HPV31 L1 VLP组(蛋白含量0.5μg,铝含量0.24 mg)、HPV33 L1 VLP组(蛋白含量0.5μg,铝含量0.24 mg)和佐剂对照组(铝含量0.24 mg),分别于0、1、3周经腹腔注射免疫小鼠,于每次免疫后1周经眼球采血,分离血清,采用假病毒中和试验检测血清中和抗体滴度。结果纯化的HPV31和HPV33 L1重组蛋白相对分子质量约为56 000,可与HPV L1通用型单克隆抗体发生特异性结合。经透射电镜及动态光散射观察,可见VLPs形成,但部分L1蛋白以五聚体的壳粒形式存在,未包装成颗粒。HPV31和33 L1 VLPs初次免疫后1周,即能诱导BALB/c小鼠产生较高滴度的血清中和抗体,且明显高于佐剂对照组(P0.05),随着免疫次数的增加,HPV31 L1 VLP组和HPV33 L1 VLP组免疫小鼠血清中和抗体滴度也相应增加。结论应用汉逊酵母可高效表达HPV31和HPV33 L1重组蛋白,并形成VLPs。用含铝佐剂的重组蛋白免疫BALB/c小鼠,具有较好的免疫原性,可作为多价重组HPV疫苗的组分抗原。
[Abstract]:Objective to express human papillomavirus (human papillomavirus,HPV) 31 and 33 L1 protein like virus granules (virus-like particles,VLPs) by using Hansenomyces cerevisiae system and to detect their immunogenicity after purification. Methods Recombinant Hansenomyces cerevisiae expressing HPV31 and HPV33 L1 protein were fermented with high density and collected. After high pressure homogenization and fragmentation, HPV31, HPV33 L1 VLPs,SDS-PAGE and Western blot were purified by affinity chromatography. The morphology of VLPs was observed by transmission electron microscope. The particle size and distribution of VLPs were analyzed by dynamic light scattering instrument. BALB/c mice were randomly divided into three groups: HPV31 L1 VLP group (0.5 渭 g protein content, Al 0.24 mg), HPV33 L1 VLP group) and adjuvant control group (0.5 渭 g protein content, 0.24 mg), HPV33 L1 VLP group) and adjuvant control group (0.5 渭 g protein content, 0.24 mg), HPV33 L1 VLP group) and adjuvant control group (0.5 渭 g protein content and 0.24 mg), HPV33 L1 VLP group respectively). Blood samples were collected from eyeball one week after each immunization and serum was isolated. Serum neutralizing antibody titer was detected by pseudovirus neutralization test. Results the relative molecular weight of the purified HPV31 and HPV33 L1 recombinant proteins was about 56000, which could specifically bind to HPV L1 universal monoclonal antibody. The formation of VLPs was observed by transmission electron microscopy and dynamic light scattering, but some L1 proteins existed in the form of shell particles of pentamer and were not packaged into granules. HPV31 and 33 L1 VLPs were not packed into granules at 1 week after primary immunization. It could induce BALB/c mice to produce high titer neutralizing antibody, and it was significantly higher than that of adjuvant control group (P0.05). With the increase of immunization times, serum neutralization antibody titer of HPV31 L1 VLP group and HPV33 L1 VLP group increased accordingly. Conclusion the recombinant protein of HPV31 and HPV33 L1 can be expressed efficiently by using Hansenia cerevisiae and VLPs. can be formed. The recombinant protein containing aluminum adjuvant was used to immunize BALB/c mice with good immunogenicity and could be used as a component antigen of multivalent recombinant HPV vaccine.
【作者单位】：北京生物制品研究所有限责任公司;北京天坛生物制品股份有限公司;
【基金】：863计划重大专项(2012AA02A401)
【分类号】：R392-33

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