新型鼠疫疫苗F1抗原原液性质的研究  

The characteristics of F1 antigen in stock solution of new type plague vaccine

作　　者：王婷 吴智远 李朋伟 张婷婷 戈钰雯 高明 刘月萍 孙田华 焦磊 王秉翔 WANG Ting;WU Zhi-yuan;LI Peng-wei;ZHANG Ting-ting;GE Yu-wen;GAO Ming;LIU Yue-ping;SUN Tian-hua;JIAO Lei;WANG Bing-xiang(The Third Research Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第三研究室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2020年第3期15-20,共6页Progress In Microbiology and Immunology

摘　　要：目的新型鼠疫疫苗包含鼠疫减毒菌EV株培养提取的天然F1抗原。研究生理氯化钠溶液中F1抗原的生物与理化性质、结构概况及聚合状态等特性,以期对F1抗原的制备、质量控制及其免疫原性研究提供理论依据。方法①过碘酸-希夫(PAS)染色法、糖蛋白碳水化合物估测试剂盒鉴定F1抗原是否为糖蛋白,蒽酮-硫酸法定量测定F1抗原中多糖含量,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术测定F1抗原单体相对分子质量并与理论值差异比对,间接证明F1抗原是否糖基化;②高效分子排阻色谱法联用多角度激光散射(HPSEC-MALLS)法研究保存于生理氯化钠溶液中F1抗原的天然聚集状态;③搜索GeneBank获得鼠疫菌F1抗原的核苷酸序列,经DNAStar软件翻译出氨基酸序列,并通过肽段覆盖率验证成熟F1抗原的氨基酸序列,以圆二色谱试验结合K2D2软件推测二级结构,MALDI-TOF-MS测定单体相对分子质量。结果①鉴定出F1抗原单体相对分子质量为15 500的蛋白质,不含有多糖成分;②F1抗原的相对分子质量分布范围在10~6~10~7之间,呈现聚集状态;③F1抗原由149个氨基酸组成,单体相对分子质量为15 500,F1抗原的二级结构以β-sheet为主。结论新型鼠疫疫苗中F1抗原为不含多糖成分的蛋白质,天然状态下F1抗原以高分子聚集态存在,F1抗原单体相对分子质量为15 500,抗原结构性质稳定,与理论结果一致。在制造、检定和免疫学研究中应参考F1抗原的这些特性。Objective One of the components in the new type plague vaccine is the natural F1 antigen which is originated from the culture of attenuated Yersinia pastis strain EV. The research of the physicochemical property, structural profile and polymerization state of F1 antigen in physiological sodium chloride solution would be helpful for its manufacture, quality control and for the understanding of its immunogenicity. Methods ① Identify whether the F1 antigen a glycoprotein or glycosylated, directly or indirectly by means of periodic acid-Schiff staining(PAS), including glycoprotein carbohydrate estimation test(GCE kit), anthrone-sulfuric acid polysaccharide quantitation examination, and Matrix-assisted Laser desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS) mass determination;② Investigation the aggregation state and polymerization of F1 antigen through detection by High Performance Size Exclusion Chromatography Multi-angle Laser Light Scattering(HPSEC-MALLS);③ Index for the nucleotide sequence of F1 antigen acquired from GeneBank and translated it as amino acid sequence by DNAStar software. To verify the amino acid sequence of mature F1 antigen with peptide coverage experiment. To predict the secondary structure by circular dichroism experiment linked with K2 D2 software. Results ① The molecular mass of F1 antigen monomer was 15 500, which was not glycoprotein or not polysaccharide attached;② The relative molecular mass of F1 antigen ranged between 1 million and 10 million, presenting a huge aggregation state;③ The mature F1 antigen composed of 149 amino acid residues, with the secondary structure mainly of β-sheet. Conclusion The invesfigation demonstrated, the F1 antigen component in the new type plague vaccine is a protein without polysaccharide. The monomer molecular mass of F1 antigen is about 15 500 but exists in high molecular mass aggregate state as native, and it is physically stable. These characteristics should be considered in the process of preparation, verification and im

关 键 词：新型鼠疫疫苗 F1抗原 性质 糖 聚集 

分 类 号：R392-33[医药卫生—免疫学]