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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(P<0.05),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。
Objective To detect human papillomavirus (HPV) 31 and 33 type-I L1 virus-like particles (VLPs) using Hansenula polymorpha system and purify to detect their immunogenicity. Methods Recombinant Hansenula engineobiotics expressing HPV31 and HPV33 L1 protein were fermented at high density. The cells were harvested and homogenized under high pressure. HPV31 and HPV33 L1 VLPs were purified by affinity chromatography. SDS-PAGE and Western blot The recombinant proteins were identified and the morphology of VLPs was observed by transmission electron microscopy. The size and distribution of VLPs were analyzed by dynamic light scattering. The BALB / c mice were randomly divided into 3 groups: HPV31 L1 VLP group (0.5μg protein content, 0.24 mg aluminum content), HPV33 L1 VLP group (protein content 0.5μg, aluminum content 0.24 mg) and adjuvant control group (aluminum content 0.24 mg). The mice were immunized intraperitoneally at 0, 1 and 3 weeks respectively. The blood was collected by eyeball one week after each immunization. Serum neutralizing antibody titers were detected by the neutralization test. Results The relative molecular mass of purified HPV31 and HPV33 L1 recombinant protein was about 56 000, which could specifically bind with HPV L1 universal monoclonal antibody. The results of transmission electron microscopy and dynamic light scattering showed that VLPs were formed, but part of the L1 protein was in the form of pentamer capsid without packing into granules. One week after the first immunization, HPV31 and 33 L1 VLPs could induce high titers of serum neutralizing antibody in BALB / c mice, which was significantly higher than that of adjuvant control group (P <0.05). With the increase of immunization times , The antibody titer of serum neutralizing antibody in mice immunized with HPV31 L1 VLP and HPV33 L1 VLP also increased correspondingly. Conclusion Hansenula polymorpha can efficiently express HPV31 and HPV33 L1 recombinant proteins and form VLPs. BALB / c mice immunized with the recombinant protein containing aluminum adjuvant have good immunogenicity and can be used as component antigens in multivalent recombinant HPV vaccines.