人巨细胞病毒UL138含多个B细胞表位病毒样颗粒的构建及鉴定  

Formation and identification of HCMV ULI38 multi- B epitope chimeric virus-like particles

作　　者：李宝青[1] 田晓娟[2] 陈晓晴[1] 王红[1] 陶洪群[1] 郑育[1] 沈贤[3] 李文姝[2] 张丽芳[2] 薛向阳[2] 

机构地区：[1]温州医科大学附属第二医院,325000 [2]温州医科大学微生物学和免疫学教研室分子病毒与疫苗研究所热带医学研究所 [3]温州医科大学附属第一医院

出　　处：《中华实验和临床病毒学杂志》2015年第6期488-492,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金（81472308,31470891）;温州市科技局项目（Y20170744）

摘　　要：目的利用毕赤酵母系统表达HPV16L1-HCMVUL138含多个B细胞表位片段的嵌合病毒样颗粒，并评价其免疫效应。方法选择富含B细胞表位的HCMVUL138 96-138片段，构建毕赤酵母真核表达质粒pPIC3．5K／HPV16L1／UL138 96-138线性化后的重组质粒电转化GSll5毕赤酵母，甲醇诱导HPVl6L1／UL13896-138蛋白表达，采用肝素亲和层析法纯化嵌合VLPs，并加以鉴定。将纯化的嵌合VLPs免疫BALB／c小鼠，通过ELISA检测免疫小鼠血清的UL138特异性抗体水平。结果经PCR、酶切和测序分析表明成功构建了pPIC3．5K／HPV16L1／UL138 96-138嵌合重组质粒。经甲醇诱导后，SDS—PAGE和WesternBlot证实重组酵母菌裂解产物存在目的蛋白。肝素亲和纯化后，透射电镜观察到了直径大约55nm的病毒样颗粒。免疫小鼠血清的ELISA结果显示，HPVl6L1．HCMVULl38B细胞表位嵌合病毒样颗粒能诱导小鼠产生较高水平的UL138特异性IgG抗体。结论以HPV16L1为载体，利用整合型重组毕赤酵母表达系统成功制备了HCMVULl38含多个B表位片段的嵌合病毒样颗粒，免疫小鼠可产生较高水平的体液免疫反应。Objective To explore the expression of HPV16 L1-UL138 multi-B epitope chimeric virus-like particles （VLPs） in Pichia pastoris expression system and further analyze its immunological effect. Method The UL13896_13s fragment rich in B-cell epitopes was selected and inserted into pPIC3. SK eukaryotic expression plasmid. After linearized with the digestion of Bgl II restriction enzyme, the pPIC3.5K/HPV16 L1/UL13896_13s recombinant plasmid was electricity transformed into GSll5 Pichia pastoris. The expression of HPV16 L1/UL13896_I3s protein was induced by methanol. The chimeric VLPs was purified by using heparin affinity chromatography and identified by Transmission Electron Microscopy. At the same time, BALB/c mice were immunized with purified VLPs. The level of specific IgG antibody were analyzed by ELISA. Results The recombinant pPIC3.5K/HPV16L1/UL13896_~3splasmid was identified by the restriction enzyme digestion, PCR and sequencing analysis. The expression of the aim protein in the recombinant yeast was verified by SDS-PAGE and Western blot after induced by methanol. When observed in the Transmission Electron Microscopy, the purified VLPs showed the diameter with about 55nm. After immunized the mice, ELISA results showed that HPV16 L1-UL138 muhi-B epitope chimeric virus-like particles can induce mice produce high levels of specific IgG antibodies. Conclusion Using the carrier of HPV16 L1, UL138 multi-B epitope chimeric VLPs was successfully expressed in the integrated recombinant Pichia pastoris expression system. At the same time, HPV16 L1-UL138 multi-B epitope chimeric VLPs could induce the high levels of humoral immune response in mice.

关 键 词：人巨细胞病毒 病毒样颗粒 疫苗 

分 类 号：R392[医药卫生—免疫学]